Stem cells from adipose tissue, and differentiated cells from said cells

ABSTRACT

The invention concerns adult multipotent human stem cells, characterized in that they have:  
     i) significant telomerase activity,  
     ii) an HLA Class I negative phenotype,  
     iii) a normal karyotype,  
     iv) a capacity to become quiescent,  
     v) a capacity for self-renewal preserved for at least 130 population doublings.

[0001] The present invention relates to multipotent human stem cellsthat can be isolated from human adipose tissue and to the use of saidcells in therapy and cosmetology. The invention also concerns a methodfor isolating said stem cells from adult human adipose tissue and to amethod for differentiating them into cells of endodermal or ectodermalor mesodermal origin. Finally, the invention relates to screeningmethods for identifying agents that are capable of exerting an effecteither on cell differentiation or on the function of differentiatedcells.

[0002] The presence of adult multipotent “stem” cells has beendemonstrated in a large number of tissues, for example the bone marrow,blood, liver, muscle, the nervous system, and in adipose tissue. Adult“stem” cells, which in theory are capable of infinite self-renewal, havegreat cell plasticity, i.e. the abilitiy to differentiate into tissuesother than those for which it was believed they were destined. Theproperties of said cells, which are similar to those of embryonic stemcells (ES), open up considerable therapeutic perspectives especially astheir~ use does not pose the problems of compatibility and ethics,encountered with ES cells.

[0003] Unfortunately, their medical application (transplantation) iscurrently extremely limited for two main reasons:

[0004] firstly, it is very difficult to isolate said cells. Indeed Stemcells are very rare in an organism and very little is currently knownabout them, in particular at a molecular level, rendering directpurification impossible. There is a method for enriching multipotentcells (a population known as “SP” for “side population”) based on thecapacity to exclude a vital stain (Goodell M A et al.(1996), J Exp Med.vol 83, 1797-1806; Zhou S et al. (2001) Nature Medecine, vol 7,1028-1034). Other methods involve positive or negative selection, basedon the presence or absence of cell markers. As an example, Internationalpatent application WO 01/11011 describes the depletion of bone marrowcells of CD45+ glycophorin A+ cells followed by culturing CD45−/GlyA−cells in the presence of growth factors. A similar method has beendescribed by Reyes et al (Blood, November 2001, vol. 98, no 9,2615-2625).

[0005] secondly, prior amplification of said cells in theundifferentiated state in vitro poses a major problem.

[0006] While many investigators have successfully demonstrated thepresence of multipotent human cells in a large number of tissues, no-onehas been able to maintain these cells in vitro in the undifferentiatedstate beyond 50 to 80 population doublings. Self-renewal capacity is adoubly important indicator: first, because of the very limited number ofstem cells naturally present in adult tissue, a quantity of stem cellssufficient for therapeutic use can only be obtained if they can bemultiplied in vitro while preserving their original characteristics.Second, the self-renewal capacity is closely related to the verydefinition of a true stem cell (which is immortal) and thus can beindirectly correlated with extended cell plasticity. Thus, it ishighly-desirable to be able to isolate multipotent cells with aself-renewal capacity that is conserved beyond 100 population doublings.

[0007] International patent application WO 01/11011 (Furcht, Verfaillieand Reyes) describes human multipotent cells isolated from bone marrow.Said cells have a normal caryotype, a negative HLA class I phenotype andcan be maintained in culture in vitro up to 40 population doublings.However, some rare cells can reach 70 population doublings. The authorsindicate that these cells can differentiate into cells of the mesodermallineage, for example into osteoblasts, chondroblasts, adipocytes andmyocytes, and also into cells of the ectodermal lineage and of theendodermal lineage. Said cells, however, are capable of a limited numberof divisions, and so the authors suggest that, to allow the productionof a large quantity of cells, a heterologous gene coding for telomeraseshould be introduced. The heterologous gene must be excised before usingin transplantation. This reversible immortalization technique remainshighly controversial, however. There is in fact a risk of malignanttransformation of cells when an exogenous telomerase activity isintroduced at a level of expression that is non-physiological and notcontrolled by the cell (Wong Jing et al., Nature, 405, June 2000,755-756). Similar studies have also been described by Reyes et al(Blood, November 2001, vol. 98, no 9, 2615-2625). Jiang et al (Nature,Advance Online Publication 20 Jun. 2002, doi:10.1098/nature00870)describe the production of multipotent mouse and rat cells which can bemaintained in culture in vitro beyond 100 population doublings. Theauthors also make reference to a human multipotent population whichcould be maintained in vitro beyond 80 population doublings. However, nosupplemental information is given on the subject of these human cells.

[0008] Qu-Petersen et al (J. Cell Biol. 157, 5, 2002, 851-864) reportthe production of multipotent murine cells from muscle tissue. Saidcells, termed “MDSC” cells (“Muscle Derived Stem Cells”), have a normalcaryotype and have a self-renewal capacity while retaining theirmultipotentiality for about 30 population doublings. They candifferentiate into cells from different lineages. However, themultipotent character disappears beyond 40 population doublings, atwhich stage the cells become senescent and die. This work stronglysuggests that said multipotent cells display immunoprivileged behavior.Indeed, injection of said cells into the muscles of dystrophic micewhich are immunologically different from those from which the MDSC cellsare derived, results in substantial muscle regeneration even in theabsence of immunosuppressive substances of the cyclosporine type.Surprisingly,. the authors observed that the transplantation did notcause infiltration of tissue by CD4⁺ and CD8⁺ lymphocytes of the graftedmouse. This tolerance is at least in part explained by the negative MHCclass I phenotype of the MDSC cells. This work thus shows that MDSCcells are not recognized by the T lymphocytes of the (immunologicallyincompatible) receiver, and suggests that said cells could be used inallo-transplantation. This study was not extended to human cells.

[0009] Two hypotheses can be proposed to explain the limitedself-renewal capacity shown by the different multipotent cells isolatedup to now:

[0010] firstly, it can be assumed that the various studies were notcarried out on true “stem” cells but rather on intermediate precursors.This hypothesis is all the more supported by on the fact that stem cellscan readily be confused with precursors in terms of plasticity. Further,contamination of the culture by precursors is facilitated by theirabundance compared with “stem” cells, but their lifetime is limited;

[0011] secondly, it is also possible to envisage that the “stem” cellscould not be maintained in vitro in the undifferentiated state as theculture conditions were unsuitable.

[0012] It should also be noted that to date, many of the methods carriedout to obtain multipotent cells use bone marrow as the cell source.However, removing cells from the bone marrow is a difficult operationinvolving risks for the patient and representing a meager source of stemcells. Thus, it is a technique that is poorly suited to the large scaleproduction of stem cells.

[0013] Several teams of investigators have therefore attempted todevelop methods that allow isolation of multipotent cells from other,more abundant tissues, which methods do not run major risks for thepatients. From this standpoint, adipose tissue a priori constitutes apromising source. However, to date, while the presence of multipotentcells has been demonstrated in human adipose tissue, the results havebeen fairly disappointing. The cell populations obtained are oftenheterogeneous and cannot be maintained in culture in vitro beyond two orthree population doublings. Further, to date, no investigators havereported the production from human adipose tissue of multipotent cellswith a negative HLA class I phenotype. This characteristic, which is notrequired for self-grafting use, becomes indispensable if cells areintended for broader therapeutic use, in particular forallo-transplantation.

[0014] For example, International patent applications WO 01/62901(Artecel Sciences Inc) and EP 1 077 254 (Zen Bio Inc) describe theproduction, from adipose tissue, of populations of stromal cells with amultipotent character. Said populations are heterogeneous and contain,inter alia, pericytes, endothelial cells and smooth muscle cells (seeErickson et al., Biochem. and Biophys. Res. Com. 290, 763-769, (2002)).Their self-renewal capacity is extremely limited and an analysis of theexpression of surface markers confirms that they are positive HLA classI. The characteristics of said cell populations are thus not compatiblewith their use in therapy.

[0015] American patent US 2002/0076400 (Katz et al.) and WO 00/53795(University of Pittsburgh and the Regents of the University ofCalifonia) also describe the production of multipotent cell populationsfrom human adipose tissue. Said cell populations can be differentiatedinto adipocytes, osteoblasts, chondrocytes and myocytes. According tothe authors, they can be maintained in culture in vitro for at least 15cell transfers without losing their multipotent character. Noinformation is given regarding the corresponding population doubling.Before subjecting the cells to successive transfers, a telomeraseactivity was detected in said population, which was heterogeneous. Thisactivity was not measured after successive transfers. No surface markeranalyses were carried out. The expression of HLA class I antigens wasthus not determined.

[0016] The present invention overcomes the disadvantages of thetechniques described above.

[0017] The present inventors have developed a method that canreproducibly isolate multipotent “stem” cells from the adipose tissue ofyoung children and multiply them in the undifferentiated state in largequantities in vitro for more than 200 population doublings. Theirtherapeutic use thus becomes possible. In its major aspect, theinvention concerns a method for producing stem cells from adipose tissueand also the use of the stem cells obtained.

[0018] In the context of the present invention the following termssignify: Self-renewal: the capacity of division without altering theinitial characteristics of the cell. stem cell: multipotent cell with ahigh self renewal capacity, with a telomerase activity and capable ofbecoming quiescent. adult stem cell: stem cell other than an embryonicstem cell deriving, for example, from a newborn, a child or an adult.multipotent or capable of differentiating into at least two cell types.multipotential: quiescent: the capacity of a cell to remain in anon-proliferating and non-senescent state.

[0019] More particularly, the invention concerns a method for producinghuman multipotent stem cells from adult tissue in particular from adultadipose tissue. In a first step, the method comprises culturing cellsderiving from a tissue sample, preferably adult adipose tissue. Othertypes of tissue which can be used include the muscle, bone marrow,liver, and nervous system. After 12 hours of culture, the cells areseparated into two sub-populations depending on their adhesion rate, afirst cell population “CA” adhering in less than 12 h, and a second cellpopulation “CS” adhering more slowly and occurring, after 12 hours ofculture, in suspension in the culture medium. The “CA” population isthen enriched until a population of cells that is capable of becomingquiescent is obtained. From this stage, intensive proliferation of stemcells of the “CA” population can then be induced.

[0020] In a preferred variant of the invention, the method for producingmultipotent human stem cells comprises the following steps:

[0021] a) enzymatic digestion of a sample of adipose tissue;

[0022] b) recovering a cell fraction that is free of adipocytes,containing all of the cell types present in the preparation obtained in(a) with the exception of adipocytes;

[0023] c) in vitro culture for at least 12 hours of the cell fractionobtained in step (b),

[0024] d) selection of two cell sub-populations termed population “CA”and population “CS”, population “CA” having an adhesion rate of lessthan 12 hours, and population CS having an adhesion rate of more than 12hours;

[0025] e) enriching population “CA” until a population of cells isobtained that are capable of becoming quiescent;

[0026] f) optionally, inducing enhanced proliferation of stem cells ofthe “CA” population, for example by adding a growth factor.

[0027]FIG. 19 shows a diagram of a preferred variant of the method ofthe invention.

[0028] Step (a) : Enzymatic digestion of a Sample of Adipose Tissue:

[0029] The enzymatic digestion step is preferably carried out bybringing the adipose tissue sample into contact with an enzymaticpreparation such as collagenase for a short period, i.e. a maximum of 10minutes, and more preferably 5 to 10 minutes, or 5 to 8 minutes. Thisallows complete dissociation of the tissue while avoiding damage tocertain cell types and thus results in better viability of all of thecell types.

[0030] Regarding the nature of the adipose tissue, it preferably derivesfrom a healthy individual, preferably a healthy young child, preferablybelow 10 years of age, for example a newborn or a child of 2 to 3 monthsto 8 years old. The child may be male or female.

[0031] The age of the donor appears to be an important point. Indeed, acertain amount of data obtained from hematopoietic “stem” cells stronglysuggests that the “stem” cells not only reduce in number with the age ofthe individual but also undergo an ageing process resulting in a loss offunctionality (Geiger H and Van Zant (2002), Nature, vol3, no 4,329-333).

[0032] If this data is extrapolated, the adipose tissue of youngchildren appears to constitute a more abundant and more functionalsource of “stem” cells than the adipose tissue of adult individuals.

[0033] The adipose tissue sample can derive from any anatomical site,but is preferably a sample of tissue of extramedullary origin, moreparticularly from the umbilical region or from the pubic region or fromthe inguinal region or from the perineal region or from the abdominalregion or from the subcutaneous region. The pubic, pre-pubic, inguinaland umbilical regions are more particularly preferred.

[0034] Step (b) : Recovery of a Cell Fraction Free of Adipocytes:

[0035] The adipose tissue that has undergone enzymatic digestion is thentreated to remove the adipocytes. An adipocyte free cell fraction isthen recovered, containing all of the cell types present in the adiposetissue (for example pre adipocytes, stem cells, endothelial cells,pericytes, mastocytes . . . ) with the exception of adipocytes.

[0036] The adipocytes can be eliminated by any appropriate means.Centrifuging is particularly effective since all of the cells ofinterest can be found in the centrifugation pellet while adipocytesfloat in the supernatant.

[0037] It is important to note that this step in the procedure iscarried out without filtering, enabling all cell types other than theadipocytes to be preserved in culture. In conventional techniques forpreparing cells from adipose tissues, filtration steps, successive orotherwise, are generally carried out, depending on the authors, beforeor after centrifuging, to eliminate waste. This step, however, risks theloss of certain cell types.

[0038] Step (c): In Vitro Culture:

[0039] The cell fraction obtained during step (b) is then cultured forat least 12 hours, preferably for 12 to 80 hours, for example 12 to 72hours.

[0040] For this step of the procedure, the cells are seeded at a densityin the range 1000 to 5000 cells/cm², for example 1000 to 3500 cells/cm².Multipotent cells are not actually present in great numbers comparedwith other cell types, and high density seeding thus ensures that eachdish contains this cell type.

[0041] The culture medium used for this step of the method is normally aDMEM type culture medium supplemented with foetal calf serum without theaddition of other growth factors. As an example, a particularly suitablemedium is: DMEM+10% decomplemented foetal calf serum+antibiotics (100U/ml penicillin, 100 μg/ml streptomycin).

[0042] The cell yield for this step varies depending on the sample: 1000to 5000 cells per mg of tissue.

[0043] Step (d): Selecting Two Cell Sub-Populations:

[0044] The step of enrichment of multipotent stem cells starts by theseparation, at the start of the culture step (c), of two cellsub-populations as a function of their adhesion rate:

[0045] a cell population CA adhering in less than 12 h

[0046] a cell population CS adhering more slowly (48 to 72 h).

[0047] After 12 hours of culture, population CS occurs in suspension inthe culture medium while population CA adheres to the dishes.

[0048] “Stem” cells are found only in sub-population CA while the secondsub population contains multipotent precursors which die after about 60population doublings. While population CS therefore cannot be used forthe production of stem cells, it can nevertheless be used for otherapplications. The CS population has the following characteristics:

[0049] i) it is multipotent,

[0050] ii) it has a negative HLA Classe I phenotype,

[0051] iii) it has a normal caryotype,

[0052] iv) its self-renewal capacity is preserved for about 40 to 60population doublings,

[0053] v) its proliferation rate is not affected by Leukemia InhibitoryFactor (LIF).

[0054] This cell population thus lends itself to therapeutic andcosmetic uses comparable to those normally known for multipotent cellsof the prior art.

[0055] The step of selection of the rapidly adhering population(population CA) is important as it allows, from the start of culture, alesser dilution of the “stem” cells with respect to the bulk of thedifferent precursor cells by carrying out an initial selection.

[0056] Step (e) Enrichment for Stem Cells:

[0057] The CA and CS populations are then cultured in identicalconditions. For population CA, this culture produces a substantialenrichment in stem cells. This enrichment step is based on the fact thatthe precursors have a lifetime that is limited compared with the stemcells (which are immortal, in theory). During the initial populationdoublings, the precursors will multiply much more rapidly than the stemcells, then they will start to die, by 50 to 80 population doublings. Atthis stage, the population is highly enriched in stem cells.

[0058] During this step, each cell transfer is carried out when thecells reach 80% confluence and high density seeding is carried out, i.e.at a density in the range 1000 to 5000 cells/cm², preferably in therange 1000 to 3500 cells/cm², and more particularly in the range 2000 to2500 cells/cm².

[0059] At each cell transfer, the cells are diluted by a maximum of 2 or3 for about 50-80 population doublings (stage at which the CA populationis highly enriched in “stem” cells and the CS population dies, whichcorresponds to the death of the precursors). This step is indispensablebased on the hypothesis that the true “stem” cell, which is quiescent inits normal state, divides more slowly than the precursor. Greaterdilution of the cells during the trypsination steps could run the riskof losing these multipotent cells in certain culture dishes.

[0060] After about 50 to 80 population doublings (for example 60population doublings), the CS population has the characteristics of asenescent population (loss of proliferative potential and loss ofmultipotentiality) and dies. In contrast, the CA population at the samestage proliferates more slowly compared with the first populationdoublings (population doubling time about 72 hours compared with aninitial mean doubling time of about 36 hours) and is capable of becomingquiescent.

[0061] The CA population can be considered to have reached quiescencewhen it exhibits the following characteristics:

[0062] spontaneous stoppage of proliferation at about 70% confluence;

[0063] confluence may be achieved at this stage in the presence of bFGFor other growth factors. A reduction in the population doubling timefrom 72 hours to about 36 hours may be observed;

[0064] the effect of bFGF or other growth factors on the doubling timeis reversible.

[0065] Further, a measurement of the endogenous X-gal activitydetermined at a pH of 6 in the CA population is negative (less than0.05%), confirming that this population is in the quiescent state ratherthan in the senescent state.

[0066] The culture medium employed for this enrichment step is typicallya medium without added growth factors, for example the culture mediumDMEM+10% decomplemented foetal calf serum+antibiotics (100 U/ml ofpenicillin, 100 μg/ml of streptomycin).

[0067] Step (f): Induction of Stem Cell Proliferation in theUndifferentiated State:

[0068] After reaching quiescence, proliferation of cells in the CApopulation is induced by trypsination and dilution of the cells in newdishes. Preferably, the cells are subjected to a trypsin treatment at80% confluence and are diluted 2 to 10 times, preferably 5 to 10 timesin identical new culture dishes.

[0069] The addition of a growth factor at this stage, for example basicfibroblast growth factor (bFGF), PDGF, EGF, NGF or SCF allows intensiveproliferation of “stem” cells of the CA population. The addition ofhuman bFGF at this stage is particularly preferred.

[0070] The addition of growth factors such as bFGF incorporated, forexample, at a concentration of about 3 to 20 ng/ml of medium, inparticular 5 to 10 ng/ml, not only halves the population doubling time(for example the population doubling time without bFGF is about 72hours, while it is about 36 hours with bFGF), but also allows thepopulation to reach confluence. Without growth factors, proliferation ofa quiescent population of stem cells of the invention can be provoked bytrypsination and dilution, but proliferation will spontaneously stop atabout 70% confluence. Since confluence is indispensable in vitro forinitiating the differentiation of many cell types, the use of growthfactors such as bFGF must be envisaged for in vitro production of thistype of differentiated cell.

[0071] This step of the method is clearly distinguished from prior artmethods. In the method of the invention, growth factors such as bFGF areonly used after obtaining a population that can become quiescent. Incontrast, bFGF is generally used from the start of culture of freshlyisolated cells ((Tsutsumi S et al., Biochemical and BiophsysicalResearch Communications 288,413-419 (2001)). This very early use of bFGFhas a peverse effect as it stimulates not only proliferation of true“stem” cells but also of all precursors. This has the result of furtherincreasing the precursor/stem cell ratio, resulting in a loss of thisrare cell type by dilution. According to the invention, an innovatingpoint is the use of bFGF when the mass of precursors has disappeared andthe population, highly enriched in “stem” cells, becomes quiescent.

[0072] Inducing proliferation of stem cells can produce large quantitiesof these multipotent cells. The cells produced can be recovered from theculture media using conventional methods.

[0073] In summary, the method of the invention comprises a number ofinnovating elements that can optimize the production of stem cells:

[0074] rapid digestion with an enzymaticc preparation such ascollagenase (step a)) which allows complete dissociation of the tissuewhile preventing damage to certain cell types;

[0075] the absence of filtering steps in step b), to avoid loss ofcertain cell types on the filters;

[0076] high density cell seeding during culture steps d) and e). Indeed,multipotent cells are in small supply compared with other cell types;

[0077] isolation of 2 sub populations “CA” and “CS”, as a function oftheir adhesion rate;

[0078] late use of a growth factor such as bFGF after the cells havebecome quiescent (step f).

[0079] In carrying out the method of the invention, the presentinventors have established a plurality of human multipotent lines fromthe adipose tissue of young children. The technique of the invention hasbeen validated for a several adipose tissue samples, for example thoseshown in Table 1 (see the Examples below).

[0080] The cells of the invention have many characteristics of stemcells, for example: the capacity to become quiescent; quiescence stopsif induced to do so with bFGF or other growth factors, resulting in anintense resumption of proliferation (reversible effect of said growthfactors secreted in vivo into the organism during damage to the body);maintenance of multipotentiality over a large number of populationdoublings; significant telomerase activity; normal karyotype.

[0081] More particularly, the cells of the invention are multipotentadult human stem cells, characterized in that they have:

[0082] i) a capacity for self-renewal that is retained over at least 80population doublings, and preferably over at least 100 populationdoublings;

[0083] ii) significant telomerase activity

[0084] iii) a negative HLA class I phenotype;

[0085] iv) a normal karyotype;

[0086] v) a capacity to become quiescent.

[0087] The self-renewal capacity of the cells of the invention ispreserved over at least 80 population doublings, preferably at least 100or 130 population doublings, and more particularly over at least 200population doublings. This means that the cells of the invention arecapable of undergoing at least 80 or 130 or 200 population doublingswithout losing their original characteristics. In other words, themultipotentiality, telomerase activity, negative HLA class I phenotype,normal caryotype and the capacity to become quiescent are retainedthroughout all of these population doublings.

[0088] The telomerase activity of the cells of the invention, which canbe measured by conventional techniques, is particularly important. Inaccordance with the definition of a “stem” cell by Watt and Hogan(Science, vol 287, February 2000), telomerase activity normally meansthat the cells obtained should be capable of infinite self-renewal.Telomerase activity is only present in embryonic cells and, in theadult, in tumour cells and “stem” cells. This activity thus confirmsthat the cells are stem cells.

[0089] The level of the endogenous telomerase activity of the cells ofthe invention preferably corresponds to at least 20%, for example 20% to50% of the telomerase activity of a reference cell line, moreparticularly 22% to 50%. The reference line is typically a transformedline having endogenous telomerase activity, such as the transformedhuman line HEK293T (Human Embryonic Kidney 293 immortalised with Tangigen). This activity can be measured at any stage. It is preferablymeasured after 30 or 40 population doublings, for example at thequiescent stage after about 60 population doublings.

[0090] Regarding the immunological characteristics of the cells of theinvention, they differ from a conventional somatic cell. These cells donot express molecules of the HLA class I system on their surface(confirmed by flow cytometry), nor do they express HLA class II on thesurface. The molecules of the HLA class I system present self and nonself peptides to CD8+cytotoxic T lymphocytes, hence their critical rolein graft rejection reactions. The absence of surface HLA class Imolecules suggests the “universal” nature of the stem cells of theinvention in transplantation. Said cells could be used forallo-transplantation with no risks of rejection by the host,independently of its genotype.

[0091] The negative HLA class I and/or class II phenotype can beanalysed by any conventional technique. It is preferably measured after30 or 40 population doublings, for example at the quiescent stage atabout 60 population doublings, or at a later stage, i.e., afterquiescence, for example at 100 or 120 population doublings. During thefirst population doublings, surface expression of HLA class I moleculesis low but significant, then disappears at later stages (for exampleafter quiescence between 50 and 80 population doublings) correspondingto the stage at which early precursors disappear.

[0092] In the context of the invention, the expression “HLA negative”means that the stem cells of the invention have a level of surfaceexpression of HLA class I molecules that cannot be detected by flowcytometry with single labeling (using a fluorochrome). Preferably, the“HLA negative” cells of the invention also have a level of surfaceexpression of HLA class II molecules that cannot be detected by flowcytometry with single labeling.

[0093] Further, thanks to a phenomenon of immunoprivileged behavior, thecells of the invention in the differentiated state most probably do notinduce a rejection reaction in a host, independently of its genotype.

[0094] The cells of the invention have a normal karyotype, confirmingthat they are not transformed.

[0095] In the normal state, the cells of the invention are quiescent(Blau H M et al, Cell, 105, 829-841, Jun. 29, (2001)) and startproliferating again in the presence of bFGF. Quiescence is a particularcharacteristic of stem cells. Beyond a certain number of populationdoublings, “non-stem” cells become senescent. The quiescent state can,in theory, be maintained indefinitely. For the cells of the invention,the inventors have maintained this state for periods of up to one year.Proliferation can then be induced by trypsination and dilution,optionally accompanied by adding a growth factor. At quiescence, thecells of the invention stop proliferating spontaneously before reachingconfluence, for example between 50% and 90% of confluence, moreparticularly between 60% and 70% of confluence.

[0096] One important characteristic of the cells of the invention istheir multipotentiality. They are capable of differentiating into atleast two cell types. More particularly, they are capable ofdifferentiating into cells of endodermal origin (for example the liver)or ectodermal origin (nerve cells: astrocytes, oligodendrocytes andneurones) or of mesodermal origin. Examples of cells of the mesodermallineage that can be cited are adipocytes, osteoblasts, myocytes,endothelial cells and chondrocytes.

[0097] It has been demonstrated that the cells of the invention, even atlate stages, are capable of differentiating into functional adipocytes(demonstrated by lipolysis, GPDH activity, adipocyte markers), intofunctional osteoblasts (demonstrated by the presence of osteoblastmarkers and calcification of the extracellular matrix), into functionalmyocytes and into endothelial cells. This differentiation can take placein vitro or in vivo.

[0098] It has been demonstrated that CA cells of the invention have, atthe single cell level, the capacity of differentiating into adipocytes,osteoblasts, myocytes and into endothelial cells, i.e. each CA cell ofthe invention is capable of differentiating into these four cell types.In contrast to adipocytes, osteoblasts and myocyte cells which belong tothe “Limb bud mesoderm”, endothelial cells derive from the visceralmesoderm. The cells of the invention are thus not limited todifferentiation into cells of the mesenchymal lineage but can moregenerally differentiate into cells of the mesodermal lineage.

[0099] The inventors have investigated the presence of markers on thestem cells of the invention. They have established that they express thetranscription factors Oct-4 and Rex-1, and the surface antigen ABCG2(ABC transporter responsible for the SP phenotype, Zhou S et al., NatureMedicine, 7, no 9, September 2001, 1028-1034). The Oct-4 and Rex-1transcription factors are expressed specifically in embryonic stem cellsof mice and humans. Oct-4 is indispensable to maintaining thepluripotentiality of mouse embryonic stem cells. It has also been shownthat Oct-4 is expressed by human embryonic stem cells.

[0100] It has also been observed that the stem cells of the invention donot react to Leukemia Inhibitory Factor (LIF), at concentrations ofabout 10 ng/ml. LIF produces no changes in morphology or proliferationof the cells. In accordance with results obtained by other investigatorswith human stem cells, in particular embryonic stem cells, it cantherefore be concluded that the cells of the invention most probably donot express the receptor for LIF (LIF-R) and are thus LIF-R negative.

[0101] Preferably, the cells of the invention, after having reachedquiescence, stably exhibit the following phenotype in vitro:

[0102] HLA class I negative,

[0103] HLA class II negative,

[0104] CD3 negative,

[0105] CD13 positive,

[0106] Oct-4 positive,

[0107] Rex-1 positive,

[0108] ABCG2 positive.

[0109] These phenotype characteristics are associated with a normalcaryotype and significant telomerase activity. Preferably, the cells arealso LIF-R negative. This phenotype is stably conserved in vitro, i.e.in the absence or presence of FGF-2 and at concentrations of foetal calfserum that may exceed 10%. The phenotype is also preserved at highseeding densities, and beyond 140 population doublings.

[0110] The doubling time for the cell populations of the inventionvaries as a function of the proportion of stem cells present. As anexample, before reaching quiescence, the doubling time is about 36 to 40hours, reflecting the presence of precursors in the CA population. Asthe stem cell proportion increases, the doubling time also increases,and reaches about 70 to 80 hours at quiescence. Stem cells divide muchmore slowly than precursors. After quiescence, adding growth factorssuch as bFGF can significantly reduce the doubling time, for example toabout 36 hours, allowing intensive and rapid production of stem cells.

[0111] The cells of the invention can be genetically modified thenselected to introduce or cause expression of a novel characteristic, forexample by ablation or modification of an endogenous gene or forexpression of a transgene such as a reporter gene or a gene theexpression product of which has therapeutic properties, under thecontrol of a suitable promoter, for example ubiquitous ortissue-specific promoter.

[0112] The expression of a transgene or DNA or RNA can be eitherconstitutive or reversibly or irreversibly inducible. The heterologousDNA or RNA of interest can be carried by any expression vector, forexample a viral vector (including retroviral vectors), inert vectors,plasmid vectors or an episomal vector.

[0113] The vectors can be introduced into the cells by transfection, forexample using chemical agents such as calcium phosphate, by lipofectionor by using a physical agent such as electroporation, micro-injection,etc . . . Said vector can be maintained in the cell either in theepsiomal form or integrated into the genome, randomly or in a targetedmanner.

[0114] Said genetically modified cells can be used in gene therapy tosupply an expression product of a heterologous gene to an individual.Thanks to the multipotent nature and HLA class I negative nature, thestem cells of the invention are particularly suitable for this type ofapplication.

[0115] When the cells of the invention are transduced or transfected bya reporter gene, they can be used to carry out a number of studies. Asan example, the plasticity of multipotent adipose tissue stem cells canbe investigated using stem cells transfected with the lacZ gene codingfor β-galactosidase (blue after staining with Xgal). These labeled cellscan be used for in vitro and in vivo experiments.

[0116] For example, in vitro, the plasticity of said cells can bestudied by co-culture experiments. In particular, the capacity of saidcells (of mesodermal origin) to differentiate into cells of endodermalorigin (for example the liver) and into cells of ectodermal origin(nerve cells: astrocytes, oligodendrocytes and neurons) can be studied.

[0117] In vivo, the labeled cells can also be transplanted into theathymic (nude) mouse which has a deficient immune system and which thuscannot reject these cells. The transplanted cells do not produce atumor. The regenerating power of said cells is only visible if suitablelesions are initially produced in the mouse to be transplanted.

[0118] The stem cells of the invention do not express surface HLA classI molecules, in contrast to the majority of somatic cells. The absenceof said proteins the immune function of which is crucial, suggests thatsaid stem cells can be transplanted universally without any rejectionreaction.

[0119] Indeed, the inventors have demonstrated that the cells of theinvention can be transplanted into immunocompetent mice without arejection reaction 6 months after transplantation.

[0120] The CA cells of the invention are thus characterized in that invivo they have an immunoprivileged behavior, i.e. they do not give riseto a rejection reaction when they are transplanted into animmunocompetent mammal (such as a mouse), even after more than 10 daysfollowing transplantation, preferably after 80 days, and more preferablyafter 6 months. Transplantation can be allogenic or xenogenic. Theabsence of a rejection reaction can be determined using techniques thatcan demonstrate the absence of lymphocyte infilitration, for exampleusing anti-CD3 antibodies or using a hematoxylin stain.

[0121] Surprisingly, it has also been shown that in vivo, the cells ofthe invention have the capacity to migrate in the undifferentiatedstate. 50 days after transplantation of CA cells of the invention intothe Anterior Tibialis of an immunocompetent mouse, the presence of saidcells was observed in the damaged tissue adjacent to the injection site.This behavior suggests that the cells of the invention can contribute,by recruitment, to restoration of a normal phenotype at anatomical sitesother than the injection site.

[0122] The invention also concerns enriched populations of multipotentcells, characterized in that they comprise the stem cells of theinvention, and in that they are free of adipocytes, fibroblasts,pre-adipocytes, endothelial cells, pericytes, mastocytes and smoothmuscle cells.

[0123] The populations of the invention are preferably entirelyhomogeneous, i.e. they contain only stem cells. More particularly, thepopulations are clonal populations.

[0124] The invention also concerns the production of differentiatedcells from the stem cells of the invention.

[0125] For example, the invention concerns the production ofdifferentiated cells of the mesodermal lineage, characterized in thatstem cells of the invention are cultivated from confluence, in thepresence of a suitable differentiation medium.

[0126] The following medium can be cited as a medium that allowsadipocyte differentiation:

[0127] DMEM medium/Ham's F12 (vol/vol, 1:1), supplemented withantibiotics, for example 100 U/ml of penicillin, 100 μg/ml ofstreptomycin,

[0128] 5 μg/ml human insulin(Sigma),

[0129] 10 μg/ml of human transferrin (Sigma),

[0130] PPARγ activator, for example 1 μM of BRL49653, or 2 μm ofCiglitazone (Biomol),

[0131] 100 to 250 μM of isobutyl-methylxanthine (IBMX)

[0132] 1 μM of dexamethasone

[0133] 0.2 nM of triiodothyronin (T3 Sigma).

[0134] 48 to 72 hours later, this medium is replaced by the same mediumcontaining no IBMX or dexamethasone.

[0135] The following medium can be cited as a medium that allowsosteoblast differentiation:

[0136] DMEM supplemented with antibiotics, for example 100 U/ml ofpenicillin, 100 μg/ml of streptomycin,

[0137] 10% of decomplemented foetal calf serum,

[0138] 0.1 μM of dexamethasone (SIGMA),

[0139] 10 mM of β-glycerophosphate (SIGMA)

[0140] 50 μg/ml of ascorbic acid (SIGMA).

[0141] The medium is replaced every 2-3 days over a period of between 15and 20 days.

[0142] The following medium can be cited as a medium that allows myocytedifferentiation:

[0143] The medium sold under the trade name PromoCell, or

[0144] DMEM medium

[0145] 2% of decomplemented foetal calf serum

[0146] antibiotics (for example 100 U/ml of penicillin, 100 μg/ml ofstreptomycin)

[0147] The medium is replaced every 2-3 days over 4 to 6 weeks.

[0148] The following medium can be cited as a medium that allowsdifferentiation into endothelial cells:

[0149] DMEM medium supplemented with antibiotics,

[0150] 10 ng/ml of human VEGF₁₂₁ (SIGMA).

[0151] For adipocyte, osteoblast and myocyte differentiation, the stemcells are normally seeded at a density of about 10 000 to 25 000cells/cm².

[0152] Prior to the differentiation step, the cells are normally seededat a density of 25000 cells/cm² in a proliferation medium (DMEMsupplemented with 10% FCS and 2.5 ng/ml of FGF-2). Two days later, theculture medium is changed in the absence of FGF-2 for 48 hours. Thecells are then maintained in a differentiation medium for 10 days.

[0153] The stem cells of the invention are particularly suitable for usein therapy or in cosmetology.

[0154] The therapeutic use of the stem cells of the invention include,inter alia, use in transplantation and in gene therapy.

[0155] For example, for use in transplantation, the cells of theinvention are multiplied in the, undifferentiated state in vitrofollowed by introducing the cells into an individual. The cells caneither be injected into the circulation or implanted into an anatomicalsite. The cells then differentiate in vivo as a function of the damagedanatomical site. As an example, intramuscular transplantation of thestem cells of the invention into an individual with muscle lesions willgive rise to muscle differentiation and regeneration. Similarly, theregeneration of adipose tissue can be envisaged by in vivodifferentiation of cells to adipocytes.

[0156] Transplantation of the cells of the invention can thus be used toregenerate tissue in vivo, for example bone tissue, adipose tissue ormuscle tissue.

[0157] If necessary, transplantation can be accompanied by implantationof a matrix that can improve tissue regeneration, for example bysupplying a physical support for the proliferation of cells or bysupplying substances such as growth factors, etc. The matrix may bebiodegradable.

[0158] In accordance with the invention, transplantation may beautologous or allogenic. The cells of the invention are particularlysuited to allo-transplantations because of their HLA class I negativenature. The cells can thus be used in any individual independently ofgenotype without risking rejection.

[0159] In a further variant, the stem cells of the invention can be usedin the differentiated state, for example as adipocytes, chondrocytes,osteoblasts, myocytes etc. In this variant, the stem cells are subjectedto differentiation in vitro followed by introduction of thedifferentiated cells into the individual.

[0160] For the therapeutic applications of the invention, the stem cellsmay or may not be genetically modified. When the cells are geneticallymodified, they can be used in gene therapy to supply an expressionproduct to a patient, for example a heterologous protein. The modifiedcells can be cultivated in vitro in the undifferentiated state thenintroduced into the recipient. Alternatively, the cells can bemultiplied in vitro in the differentiated state and then introduced tothe recipient.

[0161] The invention also concerns the implementation of surgical andtherapeutic methods using the stem cells of the invention. It alsoconcerns pharmaceutical compositions comprising the stem cells of theinvention in association with a physiologically acceptable excipient.

[0162] The stem cells of the invention can also be used for in vitroproduction of proteins, which may or may not be recombinant,particularly therapeutic proteins. In fact, the cells of the inventioncan be cultivated in vitro for at least 100, for example at least 200population doublings, and thus constitute an almost inexhaustible sourceof expression products. The proteins in question can be expressionproducts of genes endogenous to the stem cells, or alternatively, can beexpression products of heterologous genes.

[0163] The cells of the invention can also be used in screening systemsfor the identification of active agents, for example gene products,seric extracts, conditioned media, products of animal or plant origin,libraries of pharmacological agents, etc.

[0164] For example, the invention comprises a screening method foridentifying agents that can modulate the differentiation of cells intocells of a mesodermal line, characterized by:

[0165] a) culturing stem cells in accordance with the invention underconditions allowing their differentiation into cells of the mesodermallineage (for example adipocytes, osteoblasts or myocytes) in thepresence of a candidate agent,

[0166] b) comparing the differentiation of cells in the presence of thecandidate agent with differentiation in the absence of the candidateagent.

[0167] The test agent may be an agent that can enhance differentiationor an agent that can prevent or slow or reduce differentiation(anti-differentiation substance) or a substance that can modify thedifferentiation route.

[0168] The invention also comprises a screening method that can identifyagents that may have a lipolytic activity, characterized by:

[0169] a) culturing stem cells of the invention under conditionsallowing them to differentiate into adipocytes;

[0170] b) bringing the adipocytes thus obtained into contact with acandidate agent and determining the lipolytic activity of the candidateagent.

[0171] The invention also comprises a screening method that can identifyagents that may have an anti-lipolytic activity, characterized by:

[0172] a) culturing stem cells of the invention under conditionsallowing them to differentiate into adipocytes;

[0173] b) bringing the adipocytes thus obtained into contact with acandidate agent in the presence of a lipolytic agent;

[0174] c) determining the anti-lipolytic activity of the candidateagent.

[0175] The invention also comprises a screening method that can identifyagents that may have an insulin-sensitising activity, characterized by:

[0176] a) culturing stem cells of the invention under conditionsallowing them to differentiate into adipocytes;

[0177] b) bringing the adipocyte thus obtained into contact with acandidate agent;

[0178] c) determining the insulin-sensitising activity of the candidateagent compared with untreated adipocytes.

[0179] The invention also pertains to the use of stem cells incosmetology.

[0180] In so far as the stem cells of the invention can differentiateinto adipocytes, they can be used in esthetic surgery or repair surgery,for example to reduce the wrinkled appearance of the skin, to reducescars or various skin blemishes, or to carry out tissue repair. Theinvention therefore concerns the implementation of said surgical methodsusing the cells of the invention.

[0181] The cells can also be included in cosmetic compositionscomprising excipients, vehicles, solvents, colorants, fragrances,antibiotics or other products and additives that are normally used incosmetic products. The inclusion of the cells in creams, pomades,ointments, gels, various fluids, etc, allows them to be applied directlyto the skin or other tissues or phanera. Thus, the invention alsopertains to cosmetic compositions containing stem cells of the inventionin an undifferentiated state, or containing differentiated cells derivedfrom the stem cells.

KEY TO FIGURES

[0182] Differents aspects of the invention are shown in the Figures:

[0183]FIG. 1: Endogenous β-galactosidase activity of CA and CS cellsdetected at pH 6.

[0184] Xgal staining, which reveals endogenous β-galactosidase activity(signifying cell senescence) carried out at the 60 population doublingstage, corresponding to cell transfer 20 (“T20”), reveals that the CSpopulation is senescent (degree of senescence 0.415±0.025%), while theCA population is simply quiescent (degree of senescence 0.045±0.01%).

[0185]FIG. 2 : Effect of bFGF (basic Fibroblast Growth Factor) at aconcentration of 5 ng/ml of medium) on CA and CS populations at latestages, i.e. after 50 population doublings.

[0186]FIG. 2 shows the proliferation of CA±bFGF and CS±bFGF (Abscissa:days after plating at 12 000 cells/35 mm diameter dish ; Ordinate :number of cells (×1000)/dish). Only cells of the CA populationeffectively respond to bFGF. After 50 population doublings, bFGF has nosignificant effect on the CS population. These observations confirm thequiescent state of the CA population and the state of senescence in theCS population.

[0187]FIG. 3 : Morphology of CA cells at late stages (after 50population doublings) in the absence of bFGF and in the presence ofbFGF.

[0188] “W/O FGF”=without bFGF

[0189] “+FGF”=with bFGF (5 ng/ml)

[0190] “FCS”=foetal calf serum

[0191] bFGF causes a change in cell morphology. When quiescent, thecells are flat and enlarged. In the presence of bFGF, and thus in theproliferative phase, they take on a fibroblast form.

[0192] The effect of bFGF is reversible.

[0193]FIG. 4: Karyotype of Primo 2CA cells.

[0194] Primo 2 cells were karyotyped for the 2 sub populations CA andCS, with or without bFGF and at different passages. For Primo 2CA cells,the caryotypes were produced at the following stages: T21=80 populationdoublings; T23=90 population doublings; T34=130 population doublings. Inall cases, the karyotypes were normal. FIG. 4 shows an example of akaryotype for Primo 2CA cells.

[0195]FIG. 5: In vitro differentiation of cells of Primo 2CA and ofPrimo 2CS into adipocytes at early stages:

[0196] CST1: population CS at passage 1 (corresponding to 3 populationdoublings);

[0197] CST7: population CS at passage 7 (corresponding to 21 populationdoublings);

[0198] CAT5: population CA at passage 5 (corresponding to 15 populationdoublings).

[0199] Oil Red O stain.

[0200]FIG. 6: Transcriptional expression of adipogenic markers duringPrimo 2 cell differentiation. Northern Blot analysis.

[0201] J0, J7, J12=0, 7 and 12 days, respectively, after induction ofdifferentiation,

[0202] CA T14: Primo 2 CA population at passage 14 (corresponding to 42population doublings).

[0203] CS T16: Primo 2 CS population at passage 16 (corresponding to 48population doublings);

[0204]FIG. 7: In vitro differentiation into osteoblasts of CA and CScells at early passage:

[0205] CST1: CS population at passage 1 (corresponding to 3 populationdoublings);

[0206] CST7: CS population at passage 7 (corresponding to 21 populationdoublings);

[0207] CAT5: CA population at passage 5 (corresponding to 15 populationdoublings).

[0208] Alizarin Red stain.

[0209]FIG. 8: Differentiation capacity of CA cells at late passage.

[0210] A: adipocyte differentiation of Primo 2CA cells. Induction ofAdipocyte differentiation was carried out when the cells were at the T30stage (about 130 population doublings). Oil Red O stain.

[0211] B: osteoblast differentiation of Primo 2CA cells. Induction ofOsteoblast differentiation was carried out when the cells were at theT30 stage (about 130 population doublings). Alizarin Red stain.

[0212]FIG. 9: Funcionality of adipocytes and osteoblasts:Transcriptional expression of specific markers either for adipocytedifferentiation or for osteoblast differentiation.

[0213] Transcriptional expression of the markers hPPARγ-2, haP2 and hOCwere determined using RT-PCR. The cells used were i) adipocytes (lefthand side of Figure), from Primo2CA, adipocyte induction having takenplace at stage T32 (about 140 population doublings), and ii) osteoblasts(right hand side of Figure), from Primo2CA, osteoblast induction havingtaken place at stage T32 (about 140 population doublings). hPPARγ-2:human peroxisome proliferator activated receptor γ: adipocyte marker.haP2: human fatty acid binding protein: adipocyte marker, hOC: humanosteocalcin (osteoblast marker)

[0214]FIG. 10: Functionality of adipocytes: lipolysis capacity of Primo2CA cells.

[0215] Lipolyses were carried out on adipocytes obtained from Primo 2CAT32 cells (about 140 population doublings), with agonists specific fordifferent β-adrenergic receptors. FIG. 10 shows the lipolysis ratesobtained and confirms the absence of β3 adrenergic receptors.

[0216]FIG. 11: Functionality of osteoblasts: Detection of calciumassociated with the extracellular matrix.

[0217] The extracellular matrix present in culture dishes after lysis ofthe cell mat was dried then incubated with the solution from a “SIGMAcalcium detection kit”. The quantity of calcium secreted by theosteoblasts was quantifed by reading the solution using aspectrophotometer (OD 575). Osteoblast functionality was confirmed.

[0218] The quantity of calcium secreted by the osteoblasts varied as afunction of the serum batch (211707, 210407, 210811, 210812, 3903,conventional). These batches may contain non characterized cytokines,hormones or growth factors present in varying proportions.

[0219] The adipocytes did not secrete significant quantities of calcium.

[0220]FIG. 12: Morphology of Primo2CA cells as a function of the numberof population doublings

[0221] A: 40 population doublings

[0222] B: 100 population doublings: quiescent

[0223] C: 150 population doublings: quiescent

[0224] D: 150 population doublings+bFGF : proliferative phase.

[0225] bFGF causes a change in the morphology of the cells. Whenquiescent, they are flattened and enlarged. In the presence of bFGF, andthus in the proliferative phase, they take on the form of fibroblasts(see also FIG. 3).

[0226]FIG. 13 : Adipocyte differentiation capacity of Primo 1CA, Primo3CA and Primo 6CA: Adipocytes after 8 days of differentiation.

[0227] A: Primo 1: differentiation induced at 50 population doublings

[0228] B: Primo 3: differentiation induced at 40 population doublings

[0229] C: Primo 6: differentiation induced at 40 population doublings

[0230]FIG. 14: Adipocyte differentiation capacity of Primo 1CA, Primo3CA and Primo 6CA: Oil red O stain

[0231] A: Primo 1: differentiation induced at 40 population doublings

[0232] B: Primo 3: differentiation induced at 25 population doublings

[0233] C: Primo 6: differentiation induced at 25 population doublings

[0234]FIG. 15: Cell marking and flow cytometry analysis.

[0235] Demonstration of HLA Class I negative nature of Primo 2CA stemcells. Single label: FITC 1. HELA: Human tumor cells. HLA Class Ipositive. 2. SVF: Adult adipose tissue, no population doublings. HLAClass I positive 3. Primo 2CA: 120 population doublingss 4. Primo 2CS:45 population doublingss Black line: Mouse IgG: negative antibodycontrol Gray line: Anti-HLA Class I W6/32 antibodies

[0236]FIG. 16: Obtaining multipotent clones from CA cells.

[0237] Clones CA1 and CA3 were placed in a culture medium permittingdifferentiation into adipocytes and osteoblasts. The adipocytes wererevealed by staining with red oil and the osteoblasts were revealed withAlizarin red.

[0238]FIG. 17: Expression of a transgene in CA stem cells.

[0239] CA stem cells were transduced by a retrovirus expressing a genefor resistance to an antibiotic, puromycin, and the reporter gene LacZ.They were then selected in the presence of puromycin. The selected cellsall expressed the LacZ gene, revealed in situ by β-galactosidaseactivity.

[0240]FIG. 18: Expression of Oct4, Rex-1 and ABCG2 in CA stem cells:

[0241] Left hand side photos: Expression of Oct-4 and ABCG2 RNA in CAcells and clone CA1.

[0242] RNA are extracted from CA and CA1 cells and the expression ofOct-4 and ABCG2 is:

[0243] amplified by RT-PCR

[0244] then detected by hybridization.

[0245] Right hand side photos: Expression of Rex-1 transcription factorby Primo 2CA cells after 80 and 160 population doublings. Thetranscription factor Rex-1 is involved in maintaining theundifferentiated state of embryonic stem cells. The numbers “1 ”, “2”and “3” mean “⁻RT (negative control)”, “CA cells”, and “CA1cells”,respectively. The PCR conditions are:

[0246] for Oct-4: 94° C., 1 min; 57° C., 1 min; 72° C., 1 min for 45cycles.

[0247] Primers: 5′-GACAACAATGAAAATCTTCAGGAGA-3′ and5′-TTCTGGCGCCGGTTACAGAACCA-3′, internal primer5′-CACTCGGTTCTCGATACTGG-3′for a 220-bp fragment

[0248] for ABCG2: 94° C., 1 min; 60° C., 1 min; 72° C., 1 min for 31cycles, Primers: 5′-GGCCTCAGGAAGACTTATGT-3′ and5′-AAGGAGGTGGTGTAGCTGAT-3′

[0249] for Rex-1: 94° C., 1 min, 60° C. 1 min, 72° C. 1 min, Number ofcycles 31; 72° 5 min, 1 cycle Primer: 5′-CTCTCCAGTATGAACCAGG-3′ and 5′-GAAAGGATCAGAACAACAGC-3′, internal primer, 5′-GGCATTGACCTATCAGATCC-3′for a 400-bp fragment.

[0250]FIG. 19: Schematic representation of a preferred variant of themethod for producing adult stem cells from human adipose tissue.

[0251]FIG. 20: Characteristics of Primo2 CA cells in terms of surfacemarkers.

[0252] Primo2 CA cells at the 80, 120 and 160 population doubling stageswere labelled with anti-HLA I, anti-HLA-DR anti-CD3, anti-CD13antibodies previously coupled with FITC or phycoerythrin. Control=IgG;the following antibodies were used: HLA class I conjugated withfluorescein (FITC); HLA-DR (HLA class II) conjugated with phycoerythrin(PE); CD3 (marker for T lymphocytes) conjugated with PE; CD13 (markersfor stromal cells of bone marrow, endothelial cells, early progenitorsof granulocytes/monocytes and their descendance) conjugated with FITC.

[0253] Thin line: IgG control

[0254] Thick line: antibody of interest.

[0255]FIG. 21: In vitro myocyte differentiation after 4 days.

[0256] Detection by immuno-histochemistry of myogenin, an early factorin myocyte differentiation. FIG. 21 illustrates micrographs of Primo2 CAcells at 150 population doublings after 4 days in the presence ofmyocyte differentiation medium. The cells are fixed with 4%paraformaldehyde for 10 minutes at ambient temperature and permeabilizedin the presence of PBS/0.1% Triton X100 for 10 minutes; the enodogenousperoxidase activity is then blocked by incubating the cells with 3% H₂O₂for 5 minutes. The cells are then incubated with the primary antibody:anti-myogenin antibody (mouse anti-human IgG) (1:100) between 30 minutesand one hour at ambient temperature then with secondary antibody(anti-mouse IgG coupled with peroxydase).

[0257]FIG. 22: In vitro myocyte differentiation after 21 days.

[0258] After 21 days in the presence of myocyte differentiation medium,the fast twitch myosin or FT myosin, a late marker for myocytedifferentiation (intracellular marker) was detected. Detection by FACS:after detaching the Primo2CA cells (150 population doublings), they arefixed in the presence of PBS/1% formaldehyde for 15 minutes at ambienttemperature then permeablized with a digitonin solution (10 μg/ml ofPBS) for 7 to 8 minutes at ambient temperature. Antibody labeling isthen carried out using the protocol described for expression of surfacemarkers (FIG. 20). The antibody used is a mouse antibody directlyconjugated to phycoerythrin and recognizing human FT myosin.

[0259] Thin line : IgG control

[0260] Thick line: FT-myosin

[0261]FIG. 23: In vitro differentiation of endothelial cells

[0262] Detection of von Willebrandt factor (vWF), a specific marker forendothelial cells, by immuno-histochemistry. FIG. 23 illustrates theexpression of vWF by Primo2 CA cells at 150 population doublings after21 days in the presence of angiogenic medium. The cells are fixed with4% paraformaldehyde for 10 minutes at ambient temperature andpermeabilized in the presence of PBS/0.1% Triton X100 for 10 minutes;the enodogenous peroxidase activity is then blocked by incubating thecells with 3% H₂O₂ for 5 minutes. The cells are then incubated with theprimary antibody: anti-vWF (goat IgG recognizing both human, rat andmouse vWF) between 30 minutes and one hour at ambient temperature thenwith secondary antibody, anti-goat IgG coupled with peroxydase (1:100).After maintaining for 21 days in a medium composed of DMEM and hVEGF121(10 ng/ml), the Primo2CA cells express vWF.

[0263] FIGS. 24 to 27: In vivo muscle regenerating power of Primo 2CAcells after 10 days, 50 days, 80 days and 6 months transplantation inthe mdx mouse with no immunosuppressor: FIGS. 24, 25, 26 and 27illustrate co-localization of human nuclei with the muscle fibersre-expressing dystrophin 10 days (FIG. 24), 50 days (FIG. 25), 80 days(FIG. 26) and 6 months (FIG. 27) after transplantation of Primo 2CAcells into the Tibialis Anterior. This co-localization was carried outby double labelling dystrophin by immunofluorescence and human nuclei byFISH. The transplanted cells are Primo 2CA cells at 160 populationdoublings, 150 000 in number.

[0264] The dystrophin immunofluorescence detection step was carried outbefore labelling the human nuclei by FISH:

[0265] Detection of dystrophin by immunofluorescence: the musclesections are incubated for one hour with an antibody recognizing humandystrophin, previously coupled with fluorescein. The antibodies used areas follows:

[0266] either an antibody specific for human and mouse dystrophin (mouseanti-human IgG1 : NCL-DYS2 from Novocastra, directed against theC-terminal end of human and mouse dystrophin) or,

[0267] an antibody specific for human dystrophin (mouse anti-humanIgG2a: NCL-DYS3 from Novocastra, directed against the N-terminal end ofhuman dystrophin)

[0268] Detection of human nuclei by FISH: The probe used to detect thehuman nuclei is a specifc probe for human centromers (α-Satellite)coupled with digoxigenin (CP5095-DG.5, Appligene Oncor). The detectionstep consists of applying an anti-digoxigenin antibody coupled withrhodamine to slides. Before analysing the sections, the nuclei arecompletely stained using a DAPI solution (blue stain). The slides arethen observed under a fluorescence microscope (Axiophot Zeiss) with a100 watt bulb and a system of filters (Perceptive ScientificInternational). Green: dystrophin; Red: human centromers Blue: nuclei(human and murine) TA: Tibialis Anterior G: Gastrocnemius

[0269]FIG. 24 (10 days): anti-dystrophin antibody: NCL-DYS2

[0270] Left Hand Side photos: untreated Tibialis Anterior (control);

[0271] Center photos: Tibialis Anterior of a mdx mouse treated withcyclosporin, 10 days after transplantation;

[0272] Right Hand Side photos: Tibialis Anterior of an immunocompetentmouse (no cyclosporin), 10 days after transplantation;

[0273]FIG. 25 (50 days): anti-dystrophin antibodies: NCL-DYS2

[0274] Left Hand Side photos: untreated Tibialis Anterior (control);

[0275] Center photos: Tibialis Anterior of a mdx mouse treated withcyclosporin, 50 days after transplantation;

[0276] Right Hand Side photos: Gastrocnemius of an immunocompetent mdxmouse (no cyclosporin), 50 days after transplantation, in adjacent TA;

[0277]FIG. 26 (80 days): anti-dystrophin antibodies: NCL-DYS2

[0278] Left Hand Side photos: Tibialis Anterior of an immunocompetentmdx mouse (no cyclosporin) 80 days after transplantation;

[0279] Right Hand Side photos: Gastrocnemius of an immunocompetent mouse(no cyclosporin), 80 days after transplantation, in adjacent TA;

[0280]FIG. 27 (6 months): anti-dystrophin antibodies: NCL-DYS2

[0281] Left Hand Side photos: Tibialis Anterior of an immunocompetentmdx mouse (no cyclosporin), 6 months after transplantation;

[0282] Right Hand Side photos: Tibialis Anterior of an untreated mouse(reference).

[0283]FIG. 28: Demonstration by comparative immunodetection of the humanorigin of the dystrophin expressed in the myofibers of transplantedmuscle : An analysis of the presence of myofibers expressing dystrophinand the subcellular localization in the tibialis anterior 10 days aftertransplantation was carried out using the following antibodies:

[0284] (a): an antibody directed against the C-terminal end of human andmouse dystrophin (mouse anti-human IgG1: NCL-DYS2 from Novocastra,),

[0285] (b) and (c): an antibody directed against the N-terminal end ofhuman dystrophin (mouse anti-human IgG2a: NCL-DYS3 from Novocastra,),

[0286] (d) and (e): an antibody specific for mouse collagen III.

[0287] The scale bar corresponds to 15 μm in FIGS. 28(a) and 28(b), andto 1 μm in FIGS. 28(c) to (e). A star * indicates a section from thesame myofiber.

[0288] The similarity between FIGS. 28(a) and (b) indicates the humanorigin of the expressed dystrophin. The human dystrophin is locatedbeneath the sarcolemma. In contrast, the mouse collagen mi is present inthe extracellular space between the myofibers (FIGS. 28(c) to (e)).

[0289]FIG. 29: Absence of cellular and humoral immune reactions 10 daysafter transplantation of the stem cells of the invention: The existenceof any lymphocyte infiltration following transplantation of Primo 2CAcells into an immunocompetent mdx mouse was studied using hematoxylin(FIGS. 29(a), (b) and (c)), or mouse anti-CD3 antibody (FIGS. 29(a′),(b′) and (c′)).

[0290] FIGS. 29(a) and (a′): Tibialis anterior of untreated mdximmunocompetent mouse (control);

[0291] FIGS. 29 (b) and (b′): Tibialis anterior of mouse, 10 days aftertransplantation of Primo 2CA cells, 150,000 in number;

[0292] FIGS. 29 (c) and (c′): Tibialis anterior of mouse, 10 days aftertransplantation of unpurified human stromal-vascular cells isolated fromadipose tissue;

[0293] The scale bar corresponds to 50 μm in FIGS. 29(a) to (c), and to20 μm in FIGS. 29(a′) to (c′).

[0294] 10 days after transplantation of the Primo 2CA cells, nolymphocyte infiltration (CD3⁺) is observed (see FIGS. 29(b) and (b′),compared with FIGS. 29 (a) and (a′)). In contrast, transplantation ofunpurified stromal-vascular cells isolated from human adipose tissueinduced a cytotoxic and humoral reaction (FIGS. 29(c) and (c′)).

[0295]FIG. 30: Absence of cellular and humoral immune reactions 6 monthsafter transplantation of Primo 2CA cells: The existence of a possibleimmune reaction 6 months after transplantation of Primo 2CA cells into amdx immunocompetent mouse was studied by application of the sametechniques as those described for FIG. 29.

[0296] Left Hand Side photos: Tibialis Anterior of mdx immunocompetentmouse, 6 months after transplantation of Primo 2CA cells, labelling withhematoxylin;

[0297] Right Hand Side photos: Tibialis anterior of mdx immunocompetentmouse, untreated (control), labelling with hematoxylin.

[0298] The absence of infiltration by CD3+ T lymphocytes was determinedin the muscle transplanted with the Primo 2CA cells, signifying theabsence of a rejection reaction 6 months after transplantation.

[0299]FIG. 31: In vivo muscle regeneration power of Primo1CA cells andPrimo 3 CA cells 10 days after transplantation without immunosuppressorinto the mdx mouse:

[0300] The in vivo muscle regeneration capacity was evaluated byco-localisation of human nuclei with muscle fibers re-expressingdystrophin. The co-localisation technique is identical to that describedfor the Primo 2CA cells (see legends to FIGS. 24 to 27), but thetransplanted cells are Primo 1CA or Primo 3CA cells.

[0301] photo PRIMO1 : Tibialis Anterior of an mdx immunocompetent mouse(no cyclosporin), 10 days after transplantation of Primo 1CA cells at 50and at 120 population doublings, 150 000 in number. The antibody used todetect the dystrophin is an antibouy specific for human dystrophin(mouse anti-human IgG2a: NCL-DYS3 from Novocastra, directed against theN-terminal end of human dystrophin)

[0302] PRIMO3 photos: Tibialis Anterior of an mdx immunocompetent mouse(no cyclosporin), 10 days after transplantation of Primo 3CA cells at45, 80 and 110 population doublings, 150 000 in number. The antibodyused to detect the dystrophin is also NCL-DYS3 from Novocastra.

[0303] After 10 days of transplantation, a potential for muscleregeneration was visible, both for the Primo 1 CA cells and for thePrimo 3 CA cells.

[0304]FIG. 32: Absence of cellular and humoral immune reactions 10 daysafter transplantation of Primo 3 CA cells:

[0305] The existence of a possible immune reaction 10 days aftertransplantation of Primo 3CA cells in an immunocompetent mdx mouse wasstudied by applying the same techniques as those described for Primo 2CAcells (see legends to FIGS. 29 and 30).

[0306] Primo 3 photo: Tibialis anterior of mdx immunocompetent mouse, 10days after transplantation of Primo 3CA cells (110 population doublings,150 000 in number), labeling with hematoxylin.

[0307] An absence of lymphocyte infiltration is observed with the Primo3CA cells, suggesting behavior identical to that of the Primo 2CA cells.

EXAMPLES 1—Method for Obtaining and for in Vitro Expansion ofMultipotent Stem Cells from Adipose Tissue 1.1. Description of AdiposeTissue Samples Obtained

[0308] Six samples of adipose tissue were obtained from young childrenaged 1 month to 7 years; the sexes and anatomical locations weredifferent.

[0309] Table I shows the origin of each sample, the weight thereof andthe number of cells obtained using the technique described below. TABLEI Human adipose tissue samples used for the production of multipotentstem cells Number Sample Anatomical Sample of cells name Sex Agelocation weight obtained Primo 1 F  2 years 7 months Umbilical  300 mg 400 000 region Primo 2 M  5 years Pubic region  400 mg  500 000 Primo 3M  4 months Prepubic  210 mg  400 000 region Primo 4 F  7 years Inguinalregion  2.1 g 2 000 000 Primo 5 M  1 month unknown  200 mg 1 000 000Primo 6 M 18 months unknown  200 mg  350 000

[0310] The Examples below describe obtaining multipotent stem cells fromsamples Primo 1 to 6. Said stem cells were obtained by carrying out thefollowing steps:

[0311] isolating multipotent cells from the sample

[0312] in vitro enriching of the cell culture in multipotent cells

[0313] obtaining a population of quiescent stem cells

[0314] inducing intensive proliferation of stem cells

[0315] The stem cells obtained were characterized by

[0316] measurement of telomerase activity

[0317] producing Karyotypes at different passage

[0318] studying cell plasticity (differentiation into different celltypes)

[0319] determining presence or absence of cell markers

[0320] cloning stem cells

[0321] The methodology and results are described in detail below.

1.2 Method for Isolating Multipotent Cells from the Adipose Tissue ofYoung Children

[0322] After surgery, the sample is stored in DMEM medium (Dulbecco'sModified Eagle's Medium). +10% foetal calf serum, at ambienttemperature. The tissue is rinsed in PBS (Phosphate Buffer Saline) at37° C. then drained and weighed. The sample is then very finely choppedto optimize the enzymatic digestion step.

[0323] The digestion medium is composed of DMEM medium (Dulbecco'sModified Eagle's Medium) containing antibiotics (100 U/ml of penicillinand 100μg /ml of streptomycin), 2 mg/ml of collagenase (Boerhingerreference 103586) and 20 mg/ml of bovine serum albumin fraction 5 (SigmaA reference 2153). The digestion volume isa function of the tissueweight; generally, 1 ml of digestion medium for 100 to 200 mg of tissue.Digestion is carried out at 37° C. under gentle agitation. In contrastto conventional techniques for the preparation of human preadipocytes,the digestion period is very rapid, 5 to 10 minutes, which correspondsto complete dissociation of the tissue by the collagenase. Thecollagenase activity is then inhibited by adding foetal calf serum (200μl/ml of digestion medium).

[0324] The cell preparation is then centrifuged for 5 min at 1000 rpm, astep which enables the separation of the adipocytes (which float) fromother cell types contained in the adipose tissue (pre-adipocytes, stemcells, endothelial cells, pericytes, mastocytes . . . ). It is importantto note that this step in the procedure is carried out withoutfiltration, which means that all of the cell types contained in theadipose tissue are preserved (with the exception of the adipocytes).

[0325] The cell pellet obtained after centrifuging is re-suspended inthe culture medium: DMEM+decomplemented foetal calf serum+antibiotics(100 U/ml of penicillin, 100 μg/ml of streptomycin). The number of cellsobtained is counted. The cell yield varies depending on the samples 1000to 5000 cells per mg of tissue. The cells are seeded at high density,1000 to 3000 cells per cm into plastic dishes (crystalline polystyrene,Greiner).

[0326] When put into culture, two cell sub-populations are isolatedaccording to their adhesion rates. The first sub-population, designated“CA”, is constituted by cells which adhere very rapidly (less than 12h). The second sub-population, CS, is constituted by cells that adheremuch more slowly (48 to 72 h).

[0327] In practice, 12 h after starting the culture, certain cells haveadhered to the plastic. Those cells constitute the CA population. Thecells constituting the CS population are at the same moment insuspension in the culture medium. That culture medium is removed anddeposited in a new culture dish. After 72 h, the CS cells have adhered.

1.3 Enriching the Culture in Multipotent Stem Cell 1.3.1 Obtaining aPopulation of Quiescent Stem Cells

[0328] The two sub-populations, CA and CS, are maintained in culture inthe same way. Said cells, in the early stages (corresponding to about50-60 population doublings) have similar characteristics in terms ofplasticity, proliferation and morphology.

[0329] The cells are maintained in the culture medium DMEM+10%decomplemented foetal calf serum+antibiotics (100 U/ml of penicillin,100 μg/ml of streptomycin).

[0330] When the cells reach 80% confluence, they are treated withtrypsin (Trypsine-EDTA, Invitrogen) and taken up into culture in threenew dishes of identical diameter. The seeding density corresponds to1000 to 3500 cells/cm². The cells are deliberately not diluted furtherto preserve the multipotent cells, which theoretically divided moreslowly than the precursors, in all of the dishes.

[0331] The cells were maintained under these conditions until they stopproliferating. For Primo 2, the CA and CS cells stop proliferating aftercell transfer 20 (T20) corresponding to 60 population doublings.

[0332] Xgal staining (revealing the endogenous β-galactosidase activitydetected at pH 6 and demonstrating cell senescence) revealed that the CSpopulation was senescent while the CA population was simply quiescent(see FIG. 1).

[0333] For this enrichment step, different culture media were tested. Itwas observed that the stem cells of the invention did not react to“Leukemia Inhibitory Factor” (LIF) (10 ng/ml). LIF produces no change inthe morphology or proliferation of the cells. This tends to confirm thatthe cells do not express the LIF receptor (LIF-R⁻).

1.3.2 Induction of Intensive Stem Cell Proliferation

[0334] After establishing a population of quiescent CA cells, human bFGF(basic Fibroblast Growth Factor) is added to the culture mediumdescribed above in a concentration of 5 ng/ml of medium.

[0335] As indicated in FIG. 2, only cells of the CA populationeffectively respond to bFGF. In contrast, bFGF had practically no effecton the CS population at late stages (i.e. after 50 populationdoublings).

[0336] These observations confirm once again the quiescent state of theCA population and the senescent state of the CS population.

[0337] The CA cells, treated with bFGF, are subjected to trypsintreatment at 80% confluence, this time diluted 5 to 10 times in newidentical culture dishes.

[0338] Two supplemental points can be made regarding bFGF:

[0339] bFGF causes a change in the morphology of the cells. Whenquiescent, they are flattened and enlarged. In the presence of bFGF, andthus in the proliferative phase, they take on a fibroblast form (FIG. 3,FIG. 12)

[0340] further, the effect of bFGF is reversible (FIG. 3).

1.4. Freezing Cells from the Two Sub-Populations CA and CS

[0341] Cells from the two sub-populations CA and CS are frozen regularlyto constitute a stock of each cell population and to allow itsdevelopment during cell transfers to be followed. Cryoconservation doesnot change the properties of said cells.

[0342] In practice, the cells were trypsinated, centrifuged andre-suspended in a freezing medium constituted by foetal calf serumsupplemented with 10% DMSO. These cells were then placed at −20° C. for1 h then at −80° C. overnight and finally stored in liquid nitrogen at−180° C.

2. Measurement of Telomerase Activity of the Stem Cells 2.1 Methodologyfor Determining Telomerase Activity

[0343] The telomerase activity is quantified using a TeloTAGGGTelomerase PCR Elisa^(PLUS) kit (Roche).

[0344] The telomerase activity is quantified in two steps:

[0345] i) The first step is an amplification/elongation step (or TRAPassay) wherein the telomerase adds telomeric motifs (TTAGGG) to the 3′end of a biotinylated primer.

[0346] ii) The second step is detection and quantification by Elisa.

[0347] The PCR products obtained in step 1 are hybridized with aspecific primer for telomeric ends labeled with digoxigenin. Further,Elisa microplates were treated with streptavidin to immoblise theproducts via the biotin. The immobilized amplicons were detected with ananti-digoxigenin antibody conjugated with an anti-DIG-HRP and theperoxidase substrate TMB.

[0348] The intensity of the photometric reaction was estimated using anElisa microplate reader (absorbance at 450 nm with a referencewavelength of 690 nm).

[0349] The relative telomerase activity of the sample is then calculatedwith respect to the telomerase activity of a positive control (cellsfrom the HEK293 line, Human Embryonic Kidney 293).

2.2 Results of Telomerase Activity Determination

[0350] Using the “TeloTAGGG telomerase PCR Elisa Plus” kit sold byRoche, the telomerase activity present in the 2 sub-populations CA andCS of Primo2 was quantified.

[0351] A significant telomerase activity was detected in the stem cellsof the invention. For Primo 2CA (T25: cell transfer 25, corresponding toabout 100 population doublings), the telomerase activity was about 20%compared with the telomerase activity of the transformed human lineHEK293T (Human Embryonic Kidney 293 immortalized by T antigen). TheHEK293T line is used in this kit as a reference.

[0352] In contrast, no significant telomerase activity was detected inthe CS cells, for example the cells from Primo 2CS (T20) had an activityof about 5% compared with that of HEK293T.

3. Karyotype of Stem Cells

[0353] The karyotype allows observation and classification of thechromosomes present during metaphase.

3.1 Methodology for Determining Karyotype

[0354] Metaphases are obtained using conventional cytogenic techniques.After accumulating the cells in metaphase by blocking the fusorialapparatus (incubation in the presence of colchicine for 3 h), chromosomedispersion is carried out in the cytoplasm by the action of a hypotonicsolution (75 mM KCl for 40 min at 37° C.) followed by fixing with.methanol/acetic acid (3/1). The chromosomes are then identified usingRHG-banding techniques.

3.2 Results of Karvotype Determination

[0355] The cell karyotype was determined. These karyotypes were carriedout on the 2 sub-populations CA and CS in the presence or absence ofbFGF and at different passages (T21, T23 and T34 for Primo 2CA).

[0356] In all cases, the karyotypes were completely normal. Thus, thecells had not undergone any chromosomal rearrangement. FIG. 4 shows thekaryotype of Primo 2CA cells.

4. Cell Plasticity of the Stem Cells

[0357] Stem cell plasticity is evaluated using the following techniques:

4.1 Methodology for Evaluating Cell Plasticity 4.1.1. Conditions forDifferentiation into Different Cell Types

[0358] The cells are trypsinated then seeded at 20000 cells/cm². Thecells reach confluence 24 to 48 hours later. As confluence is a criticalstep for differentiation, the cells are maintained at confluence for anadditional 24 h before proceeding to differentiation (adipocytes,osteoblasts, myocytes).

[0359] i) Conditions for Differentiation into Adipocytes

[0360] The confluent cells are incubated in a DMEM/Ham's F12 (vol/vol,1:1) medium supplemented with 100 U/ml of penicillin, 100 μg/ml ofstreptomycin, 5 μg/ml of human insulin (Sigma), 10 μg/ml of humantransferrin (Sigma), 1 μM of PPAR activator (for example BRL49653), 100to 250 μM of isobutyl-methylxanthine (IBMX) and 1 μM of dexamethasone.48 to 72 hours later, this medium is replaced by the medium describedabove but containing no IBMX and dexamethasone. This differentiationmedium is replaced every 2-3 days for a period of 15 to 20 dayscorresponding to an optimum adipocyte differentiation.

[0361] ii) Conditions for Differentiation into Osteoblasts

[0362] Cells that have been confluent for 24 h are incubated with anosteoblast differentiation medium composed of DMEM, 100 U/ml ofpenicillin, 100 μg/ml of streptomycin, 10% of decomplemented foetal calfserum, 0.1 μM of dexamethasone (SIGMA), 10 mM of β-glycerophosphate(SIGMA) and 50 μM of ascorbic acid (SIGMA).

[0363] The medium is replaced every 2-3 days over a period of 15 to 20days.

[0364] iii) Conditions for Differentiation into Myocytes

[0365] Cells that have been confluent for 24 h are incubated either inDMEM medium or in PromoCell medium in the presence of 2% ofdecomphmented foetal calf serum and antibiotics (100 U/ml of penicillin,100 μg/ml of streptomycin). The medium is replaced every 2-3 days over 3to 6 weeks particularly every 3 days over 21 days.

[0366] iv) Conditions for Differentiation into Endothelial Cells

[0367] The cells are seeded at 20 000 /cm² in a DMEM medium containing10 ng/ml of human VEGF₁₂₁ (SIGMA). The differentiation medium isreplaced every 2-3 days for 21 days.

4.1.2 Stains

[0368] i) Oil Red O stain (Adipocytes: Staining of Intracellular Lipids)

[0369] After fixation in a PBS/0.25% glutaraldehyde solution, the cellsare incubated for 5 minutes at ambient temperature in a solution of OilRed O 2% (weight/volume). The cells are then washed and stored in 70%glycerol.

[0370] ii) Alizarin Red Stain (Osteoblasts: Calcification ofExtracellular Matrix)

[0371] After fixation in a PBS/0.25% glutaraldehyde solution, the cellsare incubated for 5 minutes at ambient temperature in a solution ofAlazarin Red O 1% (weight/volume). The cells are then washed with waterand stored dry.

4.1.3. Transcriptional Analysis

[0372] i) Extraction d'RNA

[0373] Cellular RNAs are extracted using Tri Reagent (Euromedex, RefTR-118)

[0374] ii) Northern Blot

[0375] 20 μg of RNA/well are deposited on an agarose gel (1.2%)/MOPS(1×)/formaldehyde (1.1M). After electrophoresis in a MOPS (1×) migrationbuffer, the RNA is transferred to a membrane of nylon Hybond N+(Amersham Pharmacia).

[0376] The membrane is then hybridized in the presence of a specificprobe labelled with ³²P [dCTP] using the Rediprime™ II Random PrimeLabelling system (Amersham Pharmacia).

[0377] iii) RT-PCR

[0378] Reverse transcription PCR reaction was carried out using aOneStep RT-PCR kit from Qiagen.

4.1.4. Analysis of the Expression of Intracellulars Markers

[0379] This technique was used to quantify the number of Primo2CA cellscapable of in vitro differentiation into myocytes. The marker that wasanalysed was fast twitch myosin ou FT myosin, a late marker formyogenesis.

[0380] After detaching the cells, they are fixed in the presence ofPBS/1% formaldehyde for 15 min at ambient temperature then permeabilizedwith a solution of digitonin (10 μg/ml of PBS) for 7 to 8 min at ambienttemperature.

[0381] Antibody labeling is then carried out, using the protocoldescribed for detecting surface markers (see Examples 7 and 11 below).The antibody used is a mouse antibody directly conjugated withphycoerythrin and recognizing human FT myosin.

4.1.5. Immunohistochemistry

[0382] The cells are fixed with 4% paraformaldehyde for 10 min atambient temperature. When the desired protein is nuclear (such asmyogenin, for example), the cells are permeabilized in the presence ofPBS/0.1% Triton X100 for 10 min. The activity of the endogenousperoxidase is then blocked by incubating the cells with 3% H₂O₂ for 5min.

[0383] The cells are then incubated with the primary antibody forbetween 30 min and 1 h at ambient temperature, then with the secondaryantibody (anti mouse IgG coupled with peroxidase (Vector Laboratories)or anti-goat IgG coupled with peroxidase (Santa Cruz Biotechnology).

[0384] The primary antibody used in our experiments, von Willebrandfactor (vWF) (goat IgG, recognizing both human, rat and mouse vWF)(Santa Cruz Biotechnology) and Myogenin (mouse anti human IgG) (SantaCruz Biotechnology), were used in a proportion of 1:100.

4.2 Results of Cell Plasticity Analysis 4.2.1. Plasticity of the TwoCell Sub-Populations CA and CS at Early Passages

[0385] At early stages (for example T1, T5, T7 corresponding to 3, 15and 21 population doublings, respectively), the CA and CS populationshave the same characteristics in terms of plasticity, morphology andproliferation.

[0386] i) Differentiation Into Adipocytes

[0387] The results for the experiments involving Oil Red O staining areshown in FIG. 5 (for Primo 2CA and Primo 2CS) and FIG. 14 for Primo 1CA(40 population doublings), Primo 3CA (25 population doublings) and Primo6CA (25 population doublings). Further, the adipocytes from Primo 1CA,Primo 3CA and Primo 6CA after 50, 40 and 40 population doublings,respectively, are shown in FIG. 13. A comparison of FIGS. 13 and 14clearly shows that the higher the number of population doublings, themore homogeneous the differentiation.

[0388] A “Northern Blot” analysis (FIG. 6) demonstrates thetranscriptional expression of adipogenic markers (aP2 and PPARγ2) duringdifferentiation: CA T14 (42 population doublings) and CS T16 (48population doublings).

[0389] ii) Differentiation Into Osteoblasts

[0390]FIG. 7 shows the differentiation of CS and CA cells intoosteoblasts. Alizarin Red stain.

4.2.2. Evolution of Cell Plasticity at Late Passages

[0391] Cells of Primo 2 CS, after transfer 20 (corresponding to about 60population doublings) become senescent. They simultaneously lose theirproliferative potential and their differentiation capacity.

[0392] In contrast, the cells of the CA population at the same stagebecome quiescent. They proliferate in the presence of bFGF and retaintheir plasticity. Said plasticity remains unchanged at transfer 40(corresponding to about 200 population doublings). FIG. 8 showsadipocyte and osteoblast differentiation for Primo2 CA T30 (about 130population doublings).

[0393] At late passages, the CA population also retains transcriptionalexpression of the different specific markers either for adipocytedifferentiation, or for osteoblast differentiation. (FIG. 9) Primo2 CAT32 (140 population doublings).

4.2.3—Capacity of Primo2 CA Cells to Differentiate into Myocytes andEndothelial Cells In Vitro

[0394] Under appropriate culture conditions, Primo 2CA cells are capableof differentiating in vitro after about 3 weeks into myocytes andendothelial cells.

[0395] i) Myocyte Differentiation

[0396] After 4 days in the presence of myocyte differentiation medium,Primo2CA cells express early markers for myocyte differentiation such asmyogenin (inmmunohistochemical labeling, cf FIG. 21). After 7 days,myogenin expression is not longer detectable.

[0397] After 21 days, 95% of the Primo2 cells cultivated in this mediumexpress a late marker for myocyte differentiation, namely Fast Twitchmyosin (intracellular labeling and FACS analysis, cf FIG. 22).

[0398] ii) Differentiation into Endothelial Cells

[0399] In contrast to adipocytes, osteoblast and myocyte cells whichbelong to the “Limb bud mesoderm”, endothelial cells derive from thevisceral mesoderm. After being maintained for 21 days in a mediumcomposed of DMEM and hVEGF121 (10 ng/ml), the Primo2 CA cells expressvon Willebrand factor, a specific marker for endothelial cells(immunohistochemistry, cf FIG. 23).

5. Characterization of Adipocyte Functionality by Enzymatic Assay 5.1Methodology for Characterizing Adipocyte Functionality 5.1.1 MeasuringGycerophosphate Dehydrogenase Activity (GPDH)

[0400] Firstly, the cells which are to have the GPDH activity measure,are lysed. The principle of the assay can be summarized in the followingscheme:

[0401] The initial rate of disappearance of NADH is determined at 340 nm(in the presence of NADH, DHAP and cell lysate), which allows thequantity of degraded substrate and hence the specific enzymatic activity(after assaying the proteins) to be calculated. The reading is made witha spectrophotometer, allowing kinetic measurements to be carried out(KONTRON Uvicon 860 thermostatted at 37° C.).

5.1.2. Lipolysis Test

[0402] This test consists of measuring the radiolabelled glycerolliberated by adipocytes in the presence of adrenergic receptor agonists.The method used is that described by Bradley D C and Kaslow H R (AnalBiochem, 1989, 180,11-16). The glycerol liberated is phosphorylated inthe presence of glycerokinase and ATP and ATP labeled with ³²P in the αposition. The residual ATP is then hydrolyzed in an acid medium at 90°C. and precipitated with ammonium molybdate and triethylamine. Theradioactivity incorporated in the form of glycerophosphate labelled with³²P is estimated by counting in a β counter and the values are expressedin pmol using a calibration curve.

5.2 Results of Characterization of Adipocyte Functionality 5.2.1.Glycerophosphate Dehydrogenase Activity (GPDH)

[0403] Primo 2CA cells (T24 in the presence of human bFGF):

[0404] After 11 days of differentiation (2 experiments)

[0405] Control: 77 nmol/min/mg of protein

[0406] In the presence of an agonist of PPARγ (BRL49653): 290nmol/min/mg of protein

[0407] After 16 days of differentiation (3 experiments)

[0408] Control: 20 nmol/min/mg of protein

[0409] In the presence of an agonist for PPARγ (BRL49653): 390nmol/min/mg of protein

[0410] Primo 2CS cells (T22 in the presence of human bFGF):

[0411] After 13 days of differentiation (3 experiments)

[0412] Control: 22 nmol/min/mg of protein

[0413] In the presence of an agonist for PPARγ (BRL49653): 30nmol/min/mg of protein

5.2.2. Lipolysis Capacity of Primo 2CA Cells

[0414] Lipolyses were carried out on Primo 2CA T32 cells with specificagonists for the different β adrenergic receptors, namely:

[0415] Isoproterenol: β1, β2 adrenergic

[0416] Dobutamine: β1adrenergic

[0417] Terbutaline: β2 adrenergic

[0418] CL316243 β3 adrenergic:

[0419] The following lipolysis rates were obtained (using a glycerolcalibration curve):

[0420] Control: 5.76 nmol/h/mg of protein

[0421] Dobutamine: 60.1 nmol/h/mg of protein

[0422] Terbutaline: 93.78 nmol/h/mg: 60.1 nmol/h/mg of protein

[0423] CL316243: 17.1 nmol/h/mg of protein

[0424] The results are shown in FIG. 10.

[0425] The lipolysis experiments show the presence of β1 and β2adrenergic receptors and the absence of β3 adrenergic receptors; theseresults are in accordance with in vivo observations (Galitzky et al;(1997) British J. Pharmacol 122: 1244-1250).

6. Characterization of Osteoblast Functionality by Detecting CalciumAssociated with the Extracellular Matrix 6.1 Methodology forCharacterizing Osteoblast Functionality

[0426] To detect the calcium secreted by osteoblasts, the cells werecultivated in the osteoblast differentiation medium described above.

[0427] After optimum differentiation, the cell mat is lysed with a 0.1 NNaOH solution for 45 min. A neutralization step is then carried out byadding 1N HCl (0.2 vol/1 vol of NaOH). The dishes in which theextracellular matrix remains are dried then incubated with the solutionfrom a “SIGMA calcium detection kit”. The quantity of calcium secretedby the osteoblasts is quantifed by measuring said solution using aspectrophotometer (DO575)

6.2. Results: Osteoblast Functionality

[0428] The functionality of the osteoblasts was demonstrated by thetechnique for detecting calcium associated with the extracellular matrix(FIG. 11).

[0429] We should also emphasize the importance of the batch of foetalcalf serum in osteoblast differentiation. This reflects the crucial rolein osteoblast differentiation of certain cytokines, hormones or growthfactors that are not characterized and which are present in varyingproportions depending on the serum batch.

7. Cell Labeling and Flow Cytometry Analysis

[0430] The HLA Class I negative nature of the stem cells of theinvention was demonstrated by flow cytometry using a conventional singlelabel system:

7.1 Single Labeling

[0431] The cells are detached then washed in PBS. After centrifuging,the cells are re-suspended and incubated with the primary antibody at aconcentration of 10 μg/ml for 30 min at 4° C. The antibodies used areeither monoclonal mouse antibodies directed against class I HLAmolecules (W6/32, Novocastra), or a mouse IgG antibody (Santa Cruz) usedas a negative control. The number of cells used for each condition is5>10⁵ to 10⁶. The following cells were used for this analysis: HeLa:Human tumor cells (positive HLA Class I: positive control). SVF: adultadipose tissue, no population doubling (positive HLA Class I) Primo 2CA:120 population doublings Primo 2CS:  45 population doublings

[0432] The cells are then washed and incubated for 20 min at 4° C. withan antibody (secondary) which is a mouse anti IgG antibody coupled withFITC (0.2 μg/10⁶ cells) (Caltag).

[0433] The cells are then washed and their fluorescence analyzed by flowcytometry (Scan FACS Becton Dickinson).

[0434] The results are shown in FIG. 15 and show that the stem cells ofthe invention (for example Primo 2CA) have a level of HLA class Imolecule expression that cannot be detected by single label flowcytometry. The stem cells of the invention are thus “HLA class Inegative”.

[0435] This flow cytometry experiment was also carried out with stemcells from Primo1CA and Primo3CA and with cells from Primo2CS (FIG. 15).In all cases, surface expression of HLA class I was negative.

8. Obtaining Multipotent Clones from CA Cells

[0436] Primo 2CA cells were seeded in a limiting dilution condition,namely ⅓of cells per well of 24 well plates, there maintained in thepresence of 10% FCS containing 5 ng/ml bFGF.

[0437] Ten days later, the clones were isolated and amplified. Theseclones preserved their undifferentiated phenotype until made todifferentiate. Their capacity for differentiation into adipocytes andosteoblasts was then demonstrated.

[0438]FIG. 16 shows two clones, CA1 and CA3, which were placed in aculture medium allowing differentiation into adipocytes and osteoblasts.The adipocytes were revealed by Red Oil stain and the osteoblasts wererevealed by Alizarn red stain. The clones analyzed by flow cytometrywere also revealed to be HLA class I and II negative.

9. Expression of a Transgene in Stem Cells

[0439] The stem cells of the invention, in particular cells from cloneCA1 (obtained as described in Example 7), were transduced at the 21population doublings stage by a retrovirus expressing a gene forantibiotic resistance, puromycin, and the reporter gene LacZ under thecontrol of an LTR promoter.

[0440] The infectious virions were produced from 293 cells stablytransfected with a PVPack-GP vector (containing gag and pol sequences)which was co-transfected with the plasmid pFB-Neo-lacZ and the vectorPVPack-VSVG expressing vector, containing the G protein of the virus forvesicular stomatitis.

[0441]FIG. 17 shows that the transduced cells subsequently selected inthe presence of puromycin all express the LacZ gene, as revealed in situby β-galactosidase activity.

10. Expression of Oct4, ABCG2 and Rex-1 in CA Stem Cells

[0442] Oct-4 is a transcription factor that is specifically expressed inmouse embryonic stem cells and is indispensable to maintaining theirpluripotentiality. It has also been shown that Oct-4 is expressed byhuman embryonic stem cells.

[0443] The transcriptional expression of Oct-4 was demonstrated in thestem cells of the invention. RNA was extracted from CA cells(homogeneous populations and clonal populations) and Oct-4 expressionwas amplified by RT-PCR then detected by hybridization. The PCRconditions were: 94° C., 1 min; 57° C., 1 min; 72° C., 1 min for 45cycles. −RT : negative control.

[0444] Similarly, transcriptional expression of ABCG2 was demonstratedin the stem cells of the invention. RNA was extracted from CA cells(homogeneous populations and clonal populations) and ABCG2 expressionwas amplified by RT-PCR then detected by hybridization. The PCRconditions for ABCG2 were: 94° C., 1 min; 60° C., 1 min; 72° C., 1 minfor 31 cycles.

[0445]FIG. 18 shows the results obtained with Primo 2CA cells attransfer 16 (48 population doublings) and for cells of the Primo 2CA1clone at transfer 5 (15 population doublings). They express Oct-4 andABCG2.

[0446] Primo 2CA cells also express the transcription factor Rex-1 (FIG.18, right hand side). The transcription factor Rex-1 is a specificmarker for mouse and human embryonic stem cells. The PCR conditions forRex-1 are as follows:

[0447] 94° C. 1 min., 60° C. 1 min., 72° C. 1 min., number of cycles 31,72° 5min 1 cycle.

11. Characerization of CA Cells of the Invention in Terms of SurfaceMarkers

[0448] Characterization of the CA cells of the invention, particular ofPrimo 2CA cells, was futher investigated in terms of surface markersusing flow cytometry.

11.1. Methodology for Analyzing the Expression of Surface Markers byFlow Cytometry

[0449] The labelling protocol was described above (see Example 7). Theantibodies used were as follows:

[0450] HLA class I conjugated with fluorescein (FITC);

[0451] HLA-DR (HLA class II) conjugated with phycoerythrin (PE);

[0452] CD-3 (marker for T lymphocytes) conjugated with PE;

[0453] CD13 (marker for stromal cells of bone marrow, endothelial cells,early progenitors of granulocytes/monocytes and of their descendance)conjugated with FITC

11.2—Results of Characterization of CA Cells of the Invention in Termsof Surface Markers

[0454] The Primo 2CA cells are surface negative for the expression ofCD3, HLA class I and HLA-DR. In contrast, they are CD13 positive (markerexpressed, inter alia, in bone marrow stromal cells (cf FIG. 20).

[0455] The absence of surface HLA class I and II molecules stronglysuggests the non immunogenicity of Primo 2CA.

[0456] It is interesting to note that Primo 2CA cells differ from thehuman bone marrow cells described by Reyes et al (Blood, November 2001).

[0457] Those cells, termed MPC, for “Mesodermal Progenitor Cells” areHLA class I and class II negative and CD13 positive only when cultivatedin low densities in a culture medium containing a low concentration offoetal calf serum (2%) with the obligatory presence of EGF and PDGF.

[0458] In contrast, cultivated in the presence of 10% foetal calf serum(concentration used to amplify the cells of the invention, in particularPrimo 2CA cells), hMPCs display the inverse phenotype, namely HLA classI and HLA-DR positive and CD13 negative. The hMPCs, cultivated in thepresence of 10% foetal calf serum or bFGF (FGF-2), lose theirproliferation potential very rapidly and die.

12. in vivo Differentiation of CA Stem Cells of the Invention intoEndothelial Cells and into Myocytes

[0459] Example 4 demonstrated that cells of the invention are capable ofdifferentiating in vitro after three weeks into endothelial and cellsand into myocytes.

[0460] The examples presented below demonstrate that the human CA cellsof the invention (particular the cells of Primo 2CA, Primo 1CA and Primo3CA) injected into the muscle of mdx mice, an animal model forDuchenne's disease in man, are capable of regenerating normal fibersafter only 10 days. Surprisingly, said cells are not rejected whentransplanted into the non immunosuppressed mouse. Said cells, by dint oftheir in vivo regeneration capacity and their absence of immunogenicity,offer a number of therapeutic perspectives in an allogenic context.

12.1 Transplantation Protocol and Analysis of in vivo Regeneration byImmunofluorescence and FISH (Fluorescence in Situ Hybridization) 12.1.1.Transplantation Protocol

[0461] To analyse the in vivo regeneration potential of the CA cells ofthe invention (more particularly cells of Primo2 CA, Primo 1CA and Primo3CA), the mdx mouse animal model (C57BL/10ScSn DMD^(mdx)/J), was used.

[0462] These mdx mice (X-chromosome—linked muscular dystrophy)constitute a good model for studying Duchenne myopathies in man as theyhave a point mutation of the gene for dystrophin (located on the Xchromosome) causing non-translation of dystrophin. In man, the absenceof dystrophin, a protein with a little known role, causes a cascade ofevents that are currently little understood, leading to the progressivedisappearance of muscle fibers and death.

[0463] The following cells were used for the in vivo muscle regenerationexperiments:

[0464] Primo 2CA cells obtained between 80 and 160 population doublings(more precisely 80, 120 and 160 population doublings);

[0465] Primo 1CA cells obtained at 50 and at 120 population doublings;.

[0466] Primo 3CA cells at 45, 80 and 110 population doublings.

[0467] In all of the experiments 3 to 4 month old mdx mice of bothgenders were used.

[0468] The left Anterior Tibialis muscle was transplanted with 150 000Primo 2CA, Primo 1CA or Primo 3CA cells taken up in a volume of 50 μl ofHBSS (Hank's Buffered Saline solution). The same volume of HBSS wasinjected into the right muscle to serve as a negative control.

[0469] To analyse the muscle regeneration potential of the cells of theinvention, in a first step we treated transplanted mice with animmunosuppressor, cyclosporin, to avoid the risk of immune rejection.The cyclosporine was administered intraperitoneally once a day in aconcentration of 10 mg/kg of animal weight/day as of transplantation.

[0470] The transplanted mdx mice simultaneously treated withcyclosporine were sacrificed after 10 days.

[0471] In parallel, to test the non immunogenicity of Primo2 CA, Primo1CA or Primo 3CA cells, the inventors used the same transplantationprotocol but on mdx mice with a normal immune system, i.e. not treatedwith cyclosporine.

[0472] The transplanted muscles and the control muscles (injected onlywith 50 μl HBSS) were recovered and frozen in isopentane then liquidnitrogen.

12.1.2 Detection of Dystrophin by Immunofluorescence

[0473] 12 μm seriated sections were prepared from the frozen anddehydrated muscles by successive passages of 10 minutes in 50, 75 and100% ethanol.

[0474] To carry out dystrophin labeling, the frozen sections were fixedin methanol/glacial acetic acid (70/30, v/v) for 15 min at ambienttemperature. After washing with PBS (phosphate buffered saline) andincubating for 30 min in a blocking solution (PBS+3% BSA (bovine serumalbumin)), the sections were incubated for one hour with a specificantibody for human dystrophin (mouse anti-human IgG2a, NovocastraNCL-DYS3), or an antibody that recognized both human, rat and mousedystrophin (mouse IgG1 NCL-DYS2, Novacastra). To reduce backgroundnoise, the antibody was first coupled with fluorescein (Alexa Fluor488), using “Zenon Alexa Fluor 488 Mouse IgG2a or IgG1 Labeling Kit”,depending on the antibodies (Molecular Probes).

[0475] The sections were then washed with PBS and then with water andanalyzed using a fluorescence microscope (Olympus BH2).

12.1.3. Detection of Human Transplanted Nuclei by FISH (Fluorescence insitu Hybridization)

[0476] The slides were dehydrated by successive passages of 2 min inbaths of ethanol in increasing concentrations (70, 80 and 100%), thendenatured at 73° C. in a 70% formamide/2×SSC solution (citrated salinesolution) for 2 min 30 s. The slides were then rehydrated (ethanol bathsin decreasing concentrations of 100, 80 and 70%) before proceeding tohybridization.

[0477] The probe used to detect human nuclei was a specific probe forall human centromers (α-Satellite) coupled with digoxigenin(CP5095-DG.5, Appligene Oncor).

[0478] The probe, initially dilated in the hybridization buffer HybrisolVI (Appligene Oncor) (1 μl of probe for 10 μl of buffer), was denaturedfor 5 min at 72° C. then deposited on the slides. The hybridization stepwas carried out at 37° C. in a moist chamber for 12 h.

[0479] The slides were then washed: 1 wash in 50% formamide/2×SSC at 43°C. for 15 min followed by washing in 2×SSC at 37° C. for 8 minutes.

[0480] The final step was a detection step consisting of applying to theslides an anti-digoxigenin antibody coupled with rhodamine (AppligeneOncor) (5 min in the dark).

[0481] Before analyzing the sections, the nuclei were stained completelyusing a DAPI solution (blue stain).

[0482] The slides were then observed under a fluorescence microscope(Axiophot Zeiss) with a 100 watt lamp and a filter system (PerceptiveScientific International).

12.1.4. Double Immnunofluorescence Labeling/FISH

[0483] In order to co-localize human nuclei with muscle fibersre-expressing dystrophin, the inventors carried out double labeling(dystrophin/human nuclei). To this end, the dystrophin detection stepwas carried out before labeling the human nuclei by FISH.

12.2—Results: Muscle Regeneration Capacity and Non Immunogenicity of CACells of the Invention in vivo 12.2.1 Muscle Regeneration Capacity invivo

[0484] Many studies regarding the multipotentiality of adult stem cellsin vivo have been questioned, in particular after demonstrating thefusion power of said cells (Terada et al, Nature 2002 ; Wurmser et al,Nature, 2002 and Ying et al, Nature, 2002). Further, certain studiessuggest that the capacity of said cells to express specific markers forthe tissue into which they have been transplanted is an extremely rareevent (Wagers et al, Science,2002; Morshead et al, Nature,. 2002).

[0485] To avoid the problems mentioned above, mdx mice, an animal modelfor Duchenne's disease, were used. These mice are deficient indystrophin (a point mutation in the gene). (In reality, there exists asmall number of reverting fibers that re-express dystrophin but thepercentage of those fibers does not exceed 1% (Hoffman et al., J NeurolSci, 1990; Gillis, J Muscle Research and Cell Motility, 1999) ).

[0486] Firstly, to avoid any rejection problems, the mdx mice weretreated with an immunosuppressor, cyclosporin.

[0487] Injection of a small number of Primo 2CA cells (100000 to 150000cells) into the Tibialis Anterior muscle resulted in restoration ofdystrophin in about 50% of the fibers in only 10 days. These positivefibers were collected in clusters (corresponding to the injectionpoint).

[0488] In agreement with the above results, using FISH, human nucleiwith muscle fibers positive for dystrophin (cf FIGS. 24 and 25) could belocated.

[0489] Similar results were obtained for Primo 2CA cells between 60 and160 population doublings, treated or not treated with bFGF (FGF-2).Similar results were obtained with the CA1 clone derived from the Primo2CA population.

12.2.2 Non Immunogenicity of CA Cells in vivo

[0490] In contrast to the majority of somatic cells, the CA cells of theinvention are free of HLA class I markers (see Example 7) and surfaceHLA class II markers (Example 11). This extremely rare phenotypestrongly suggests the non-immunogenicity of the CA cells of theinvention.

[0491] To validate the non-immunogenicity of these cells in vivo, anexperimental approach similar to that decribed in section 12.2.1 wasused, except that non immunosuppressed mdx mice were used.

[0492] After 10 days of transplantation, the muscle of the transplantedand non-immunouppressed mice re-expresses dystrophin levels comparablewith that of mice treated with cyclosporin. A muscle regenerationpotential was observed, both for Primo 2CA cells and for Primo 1CA cellsand Primo 3CA cells in the absence of an immunosuppressor (see FIG. 24for Primo 2CA cells and FIG. 31 for Primo 1CA cells and Primo 3CAcells).

[0493] After 50 days of transplantation, in the absence ofimmunosuppressor, the number of muscle fibers positive for dystrophincontinued to increase in the injected Tibialis Anterior but fibers werealso found in other muscles such as the gastrocnemius (cf FIG. 25).These results strongly suggest that Primo 2CA cells are capable not onlyof regenerating muscle at the injection point but also of migrating torepair the surrounding muscles. An increase in the percentage ofperipheral nuclei of human origin within the fibers and a reduction inthe percentage of central nuclei was observed 10 to 50 days aftertransplantation (73% a 85%, and 27% a 15% respectively), indicating thatthe injected cells participate in terminal differentiation of myocytes.

[0494] Using FISH, the human nuclei were still present and co-localisedwith the dystrophin-positive fibers. Further, the localization of acertain number of human nuclei at the outer periphery of the positivefibers suggests the presence of human satellite cells and/or ofendothelial cells of human origin.

[0495] By comparison, transplantation of a large quantity of humanmyoblasts (4 million) into non-immunosuppressed mice resulted incomplete rejection after one month (Huard et al, Muscle and Nerve, 1994)and thus no muscle regeneration.

[0496] After 80 days transplantation, the number of muscle fiberspositive for dystropin continued to increase in the injected muscle, andfibers were still found in the gastrocnemius cf FIG. 26).

[0497] After 6 months transplantation, more than 80% of the fibersexpressed human dystrophin as opposed to 50% at earlier times (FIG. 27).Further, within the fiber, we determined much more regular expression ofdystrophin compared with earlier transplantation times in whichdystrophin expression was irregular in the same fiber. Further, thetransplanted muscle exhibited a substantial improvement in fibermorphology (more regular and an absence of necrosis, an importantprocess in mdx mice of this age).

[0498] These observations indicate that the transplanted cells do notgive rise to any rejection reaction in the immunocompetent mouse. Thenon-immunogenic nature of the transplanted cells has been demonstratedusing hematoxylin stain. No rejection reaction (absence of infiltrationby CD3+ T lymphocytes) was observed after 10 days (FIG. 29), 50 days, 80days (results not shown) or after 6 months transplantation of Primo 2CAcells (see FIG. 30). Similarly, the absence of lymphocyte infiltrationcould also be observed 10 days after transplantation of Primo 1CA andPrimo 3CA cells, confirming the absence of a rejection reaction. Thesame results were obtained with Primo 1CA cells. The immunoprivilegedbehavior of Primo 1CA cells and Primo 3CA cells was thus identical tothat observed with Primo 2CA. These results were also confirmed using ananti-CD3 antibody, demonstrating the absence of lymphocyte infiltration.

[0499] In contrast, transplantation of unpurified human stromal-vascularcells isolated from human adipose tissue induced a cytotoxic and humoralimmune reaction (FIGS. 29(c) and (c′)).

[0500] In conclusion, after six months transplantation, the cells causeda substantial improvement in the transplanted muscle with a highpercentage of fibers expressing human dystrophin and an absence ofnecrosis which is observed in untreated mdx mice of the same age; thiswas in the absence of an immunosuppressor.

[0501] The human origin of the dystrophin expressed in the myofibers ofthe transplanted muscle has been demonstrated by comparativeimmunodetection, using an antibody specific for human dystrophin(directed against the N-terminal end of human dystrophin: mouseanti-human IgG2a: NCL-DYS3 from Novocastra,), and an antibody capable ofrecognizing both human dystrophin and murine dystrophin (directedagainst the C-terminal end of human and murine dystrophin: mouseanti-human IgG1 : NCL-DYS2 from Novocastra,).

[0502] The results of this comparative immunodetection are shown in FIG.28. The presence of myofibers expressing dystrophin and the subcellularlocation in the Tibialis Anterior 10 days after transplantation isvisible. The similarity between FIGS. 28(a) and (b) indicates the humanorigin of the expressed dystrophin. The human dystrophin is locatedbeneath the sarcolemma. In contrast, mouse collagen imi is present inthe extracellular space between the myofibers (FIGS. 28(c) to (e)).

[0503] The mechanisms involved in the tolerance of the CA cells of theinvention in a xenogenic context, i.e. in an organism that isimmmunologically very different (mdx mouse) still have to be elucidated.

[0504] However, it may be supposed that a certain number of cellslocated at the outer periphery of the muscle fibers play a key role inthis tolerance. These cells may play a local immunosuppression role bysynthesizing immunosuppressive factors, for example anti-inflammatorytype Th2 cytokines such as IL10 and/or by expressing surface proteinsleading to the absence of recognition by alloreactive lymphocytes of thehost (Jorgensen et al, Engineering mesenchymal stem cells forimmunotherapy, Gene Therapy 10, 928-931 (2003)).

[0505] These cells can also induce generalized tolerance by re-educatingthe host's immune system. The presence of human CA cells in the thymusand spleen of the host reinforces this hypothesis (Fändrich F et al,“Preimplantation-stage stem cells induce long-term allogeneic graftacceptance without supplementary host conditioning”, Nat. Med 8, 171-178(2002).

[0506] These results demonstrate the immunoprivilege of the human CAcells of the invention which are capable of regenerating muscle withoutbeing rejected. These cells thus offer many prospects for cell therapiesin allotransplantation. In particular, for genetic diseases such asmyopathies where autotransplantations are impossible, the use of cellssimilar to Primo 2CA would constitute a good therapeutic alternative.

1 8 1 25 DNA artificial sequence primer 1 gacaacaatg aaaatcttca ggaga 252 23 DNA artificial sequence primer 2 ttctggcgcc ggttacagaa cca 23 3 20DNA artificial sequence primer 3 cactcggttc tcgatactgg 20 4 20 DNAartificial sequence primer 4 ggcctcagga agacttatgt 20 5 20 DNAartificial sequence primer 5 aaggaggtgg tgtagctgat 20 6 19 DNAartificial sequence primer 6 ctctccagta tgaaccagg 19 7 20 DNA artificialsequence primer 7 gaaaggatca gaacaacagc 20 8 20 DNA artificial sequenceprimer 8 ggcattgacc tatcagatcc 20

1. An adult multipotent human stem cell, characterized in that it has:i) significant telomerase activity, ii) an HLA Class I negativephenotype, iii) a normal karyotype, iv) a capacity to become quiescent,v) a capacity for self-renewal preserved for at least 130 populationdoublings.
 2. Stem cell according to claim 1, characterized in that ithas a self-renewal capacity preserved for at least 200 populationdoublings.
 3. Stem cell according to claim 1 or claim 2, characterizedin that it can be isolated from human adipose tissue.
 4. Stem cellaccording to any one of the preceding claims, characterized in that itcan differentiate into a cell of endodermal, ectodermal or mesodermalorigin.
 5. Stem cell according to claim 4, characterized in that it iscapable of differentiating into an adipocyte, osteoblast, myocyte,chondrocyte or endothelial cell.
 6. Stem cell according to any one ofthe preceding claims, characterized in that it has a telomerase activitycorresponding to at least 20% of the telomerase activity of a referencecell line.
 7. Stem cell according to any one of the preceding claims,characterized in that it expresses the transcription factor Oct-4 and/orRex-1.
 8. Stem cell according to any one of the preceding claims,characterized in that it can express at least one transgene.
 9. Cellpopulation comprising a plurality of cells according to any one ofclaims 1 or 51 to 54, characterized in that it is free of adipocytes,fibroblasts, preadipocytes, endothelial cells, pericytes, mastocytes,and smooth muscle cells.
 10. Cell population according to claim 9,characterized in that it is clonal.
 11. Cell population according to anyone of claims 9 or claim 10, characterized in that it becomes quiescentafter about 60 population doublings.
 12. Cell population according toclaim 11, characterized in that it is capable of proliferating in thepresence of growth factors such as basic fibroblast growth factor(bFGF), PDGF, EGF, NGF, SCF.
 13. A method for obtaining multipotenthuman stem cells comprising the following steps: culturing cells from ahuman tissue sample, in particular human adipose tissue, selecting twocell sub-populations termed a “CA” population and “CS” population, the“CA” population having an adhesion rate of less than 12 hours, and the“CS” population having an adhesion rate of more than 12 hours, enrichingthe “CA” population until a quiescent cell population is obtained,inducing proliferation of stem cells of the “CA” population.
 14. Amethod according to claim 13, comprising the following steps: a)enzymatic digestion of a sample of adipose tissue; b) recovering a cellfraction that is free of adipocytes, containing all of the cell typespresent in the preparation obtained in (a) with the exception ofadipocytes; c) carrying out in vitro culture of the cell fractionobtained in step (b) for at least 12 hours; d) selecting two cellsub-populations, “CA” and “CS”; e) enriching population “CA” until apopulation of cells is obtained that is capable of entering a quiescentstate, f) optionally, inducing proliferation of stem cells ofpopulation“CA”.
 15. A method according to claim 13 or claim 14,characterized in that the adipose tissue sample derives from a healthychild under 10 years of age.
 16. A method according to any one of claims13 to 15, characterized in that the adipose tissue sample is a sample ofextramedullary tissue derived, for example, from the umbilical region orfrom the pubic region or from the inguinal region or from the perinealregion or from the abdominal region or from the subcutaneous region. 17.Method according to claim 13 or claim 14, characterized in that theproliferation induced in step (f) is an intensive proliferation inducedby adding a growth factor.
 18. Method according to claim 14,characterized in that enzymatic digestion in step (a) is carried out bybringing the adipose tissue sample into contact with a collagenasepreparation for a maximum period of 10 minutes.
 19. Method according toclaim 14, characterized in that the cell fraction that is free ofadipocytes is obtained by carrying out an adipocyte elimination step,for example by centrifugation.
 20. Method according to claim 14,characterized in that the cell fraction that is cultured in step (c)does not undergo any filtration steps before culturing.
 21. Methodaccording to claim 14, characterized in that the culture step (c) iscarried out in a culture medium supplemented with foetal calf serumwithout the addition of other growth factors.
 22. Method according toclaim 14, characterized in that during culture step (e), cell transferis carried out when the cells reach 80% confluence, transfer beingcarried out at a seeding density of about 1000 to 3500 cells/cm². 23.Method according to claim 13 or claim 14, characterized in that the “CA”population becomes quiescent after about 60 population doublings. 24.Method according to claim 15, characterized in that the growth factoremployed during step (f) is selected from bFGF, PDGF, EGF, NGF and SCF.25. Stem cells obtainable by carrying out the method according to anyone of claims 13 to
 24. 26. Stem cells according to any one of claims 1to 12 or 25, for use in therapy.
 27. Stem cells according to claim 26,characterized in that the therapy comprises transplantation of cellsinto an individual followed by cell differentiation and tissueregeneration in vivo.
 28. Stem cells according to claim 26,characterized in that transplantation is allogenic.
 29. Use of a cellaccording to any one of claims 1 to 8 or 25, or a cell populationaccording to any one of claims 9 to 12, for the production of atherapeutic product for in vivo tissue regeneration.
 30. Use accordingto claim 29, characterized in that the tissue is bone tissue.
 31. Useaccording to claim 29, characterized in that the tissue is adiposetissue.
 32. Use according to claim 29, characterized in that the tissueis a muscle or endothelial tissue.
 33. A method for producingdifferentiated cells of the mesodermal lineage, characterized in thatstem cells according to any one of claims 1 to 13 or 25 are cultivatedfrom confluence in the presence of a differentiation medium.
 34. Methodaccording to claim 33, characterized in that the stem cells are seededat a density of about 10 000 to 25 000 cells/cm².
 35. Method accordingto claim 33 or claim 34, characterized in that the culture medium is amedium allowing differentiation into adipocytes.
 36. Method according toclaim 33 or claim 34, characterized in that the culture medium is amedium allowing differentiation into osteoblasts.
 37. Method accordingto claim 33 or claim 34, characterized in that the culture medium is amedium allowing differentiation into myocytes, or an angiogenic medium.38. A screening method to identify agents that can modulate thedifferentiation of cells into cells of the mesodermal lineage,characterized by: a) culturing stem cells according to any one of claims1 to 13 or 25 under conditions that allow their differentiation intocells of the mesodermal lineage, in the presence of a candidate agent;b) comparing the differentiation of cells in the presence of a candidateagent with differentiation in the absence of the candidate agent. 39.Method according to claim 38, characterized in that the cultureconditions allow differentiation into adipocytes.
 40. Method accordingto claim 38, characterized in that the culture conditions allowdifferentiation into osteoblasts.
 41. Method according to claim 38,characterized in that the culture conditions allow differentiation intomyocytes.
 42. Method according to claim 38, characterized in that theagent that can modulate differentiation is an anti-differentiationsubstance.
 43. A screening method for identifying agents that may have alipolytic activity, characterized by: a) culturing stem cells accordingto any one of claims 1 to 13 or 25 under conditions allowing theirdifferentiation into adipocytes, b) bringing the adipocytes thusobtained into contact with a candidate agent, c) evaluating thelipolytic activity of the candidate agent.
 44. A screening method foridentifying agents that may have an anti-lipolytic activity,characterized by: a) culturing stem cells according to any one of claims1 to 13 or 25 under conditions allowing their differentiation intoadipocytes, b) bringing the adipocytes thus obtained into contact with acandidate agent, in the presence of a lipolytic agent, c) evaluating theanti-lipolytic activity of the candidate agent.
 45. A screening methodfor identifying agents that may have an insuline-sensitizing activity,characterized by: a) culturing stem cells according to any one of claims1 to 13 or 25 under conditions allowing their differentiation intoadipocytes, b) bringing the adipocytes obtained into contact with acandidate agent, c) evaluating the insuline-sensitizing activity of thecandidate agent.
 46. Use of stem cells according to any one of claims 1to 13 or 25 in cosmetics.
 47. A cosmetic composition comprising aplurality of cells according to any one of claims 1 to 13 or 25, inassociation with an excipient, vehicle, solvent, colorant, fragrance,antibiotic or other additives acceptable in cosmetic products.
 48. Apharmaceutical composition comprising a plurality of cells according toany one of claims 1 to 13 or 25, in association with a physiologicallyacceptable excipient.
 49. An adult multipotent human cell, termed a “CS”cell, characterized in that i) it has an HLA Class I negative phenotype,ii) it has a normal karyotype, iii) it has a self-renewal capacity thatis preserved for about 40 to 60 population doublings, iv) it is notcapable of becoming quiescent, v) its proliferation rate is not affectedby LIF.
 50. A multipotent human cell population termed a “CS” populationcomprising a plurality of cells according to claim
 49. 51. Stem cellaccording to any one of claims 1 to 8, characterized in that it has thefollowing phenotype: HLA class I negative; HLA class II negative; CD3negative; CD13 positive;
 52. Stem cell according to any one of claims 1to 8 or 51, characterized in that it has a CD 13 positive phenotype inthe presence of 10% foetal calf serum.
 53. An adult multipotent humanstem cell, characterized in that after reaching quiescence, it stablyexhibits the following phenotype in vitro: HLA class I negative, HLAclass II negative, CD3 negative, CD13 positive, LIF-R negative, Oct-4positive, Rex-1 positive, ABCG2 positive, and in that it has a normalkaryotype and significant telomerase activity.
 54. Cell according toclaim 53, characterized in that it has immunoprivileged behavior in vivoand a capacity to migrate in the undifferentiated state.